43 results on '"Ruben A. Aguilar"'
Search Results
2. Heterogeneity in Lowe Syndrome: Mutations Affecting the Phosphatase Domain of OCRL1 Differ in Impact on Enzymatic Activity and Severity of Cellular Phenotypes
- Author
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Jennifer J. Lee, Swetha Ramadesikan, Adrianna F. Black, Charles Christoffer, Andres F. Pacheco Pacheco, Sneha Subramanian, Claudia B. Hanna, Gillian Barth, Cynthia V. Stauffacher, Daisuke Kihara, and Ruben Claudio Aguilar
- Subjects
rare genetic disease ,Lowe syndrome ,OCRL1 ,phosphatase activity ,cellular phenotypes ,Microbiology ,QR1-502 - Abstract
Lowe Syndrome (LS) is a condition due to mutations in the OCRL1 gene, characterized by congenital cataracts, intellectual disability, and kidney malfunction. Unfortunately, patients succumb to renal failure after adolescence. This study is centered in investigating the biochemical and phenotypic impact of patient’s OCRL1 variants (OCRL1VAR). Specifically, we tested the hypothesis that some OCRL1VAR are stabilized in a non-functional conformation by focusing on missense mutations affecting the phosphatase domain, but not changing residues involved in binding/catalysis. The pathogenic and conformational characteristics of the selected variants were evaluated in silico and our results revealed some OCRL1VAR to be benign, while others are pathogenic. Then we proceeded to monitor the enzymatic activity and function in kidney cells of the different OCRL1VAR. Based on their enzymatic activity and presence/absence of phenotypes, the variants segregated into two categories that also correlated with the severity of the condition they induce. Overall, these two groups mapped to opposite sides of the phosphatase domain. In summary, our findings highlight that not every mutation affecting the catalytic domain impairs OCRL1′s enzymatic activity. Importantly, data support the inactive-conformation hypothesis. Finally, our results contribute to establishing the molecular and structural basis for the observed heterogeneity in severity/symptomatology displayed by patients.
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- 2023
- Full Text
- View/download PDF
3. Worldwide Prevalence of the Lingual Canal in Mandibular Incisors – A Multi-Center Cross-Sectional Study with Meta-Analysis
- Author
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Jorge N.R. Martins, Marco A. Versiani, Pablo Ensinas, Francis Chan, Narin Babayeva, Murilo von Zuben, Luiza Berti, Ernest W.N. Lam, Marcia Antúnez, Fan Pei, Catalina Mendez de la Espriella, Walter Vargas, Juan Carlos Izquierdo Camacho, Moataz-Bellah A.M. Alkhawas, Tiago Pimentel, Fábio Santiago, Hans Willi Herrmann, Antonis Chaniotis, Gergely Benyocs, Magnús F. Ragnarsson, Jojo Kottoor, Avi Shemesh, Raffaella Castagnola, Sriteja Tummala, Satoru Matsunaga, Arina Maksimova, Hani Ounsi, Abhishek Parolia, Ruben Rosas Aguilar, Olabisi H. Oderinu, Muhammad Nazeer, Carlos Heilborn, Christian Nole, Sergiu Nicola, Elena Lipatova, Hussam Alfawaz, Hussein C. Seedat, Seok Woo Chang, Jose Antonio Gonzalez, Zaher Altaki, Danuchit Banomyong, Ali Keles, Iliana Modyeievsky, Adam Monroe, Carlos Boveda, Emmanuel J.N.L. Silva, Michael Solomonov, and Joe Ben Itzhak
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General Dentistry - Published
- 2023
4. Adaptor Proteins: Inter-Organelle Traffic Controllers
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Kayalvizhi Madhivanan, Wen-Chieh Hsieh, McKeith Pearson, and Ruben C. Aguilar
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- 2023
5. Kidney-differentiated cells derived from Lowe Syndrome patient's iPSCs show ciliogenesis defects and Six2 retention at the Golgi complex.
- Author
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Wen-Chieh Hsieh, Swetha Ramadesikan, Donna Fekete, and Ruben Claudio Aguilar
- Subjects
Medicine ,Science - Abstract
Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the OCRL1 gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient's cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs) from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, OCRL1 KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2's role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.
- Published
- 2018
- Full Text
- View/download PDF
6. The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution
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Orit Rozenblatt-Rosen, Aviv Regev, Philipp Oberdoerffer, Tal Nawy, Anna Hupalowska, Jennifer E. Rood, Orr Ashenberg, Ethan Cerami, Robert J. Coffey, Emek Demir, Li Ding, Edward D. Esplin, James M. Ford, Jeremy Goecks, Sharmistha Ghosh, Joe W. Gray, Justin Guinney, Sean E. Hanlon, Shannon K. Hughes, E. Shelley Hwang, Christine A. Iacobuzio-Donahue, Judit Jané-Valbuena, Bruce E. Johnson, Ken S. Lau, Tracy Lively, Sarah A. Mazzilli, Dana Pe’er, Sandro Santagata, Alex K. Shalek, Denis Schapiro, Michael P. Snyder, Peter K. Sorger, Avrum E. Spira, Sudhir Srivastava, Kai Tan, Robert B. West, Elizabeth H. Williams, Denise Aberle, Samuel I. Achilefu, Foluso O. Ademuyiwa, Andrew C. Adey, Rebecca L. Aft, Rachana Agarwal, Ruben A. Aguilar, Fatemeh Alikarami, Viola Allaj, Christopher Amos, Robert A. Anders, Michael R. Angelo, Kristen Anton, Jon C. Aster, Ozgun Babur, Amir Bahmani, Akshay Balsubramani, David Barrett, Jennifer Beane, Diane E. Bender, Kathrin Bernt, Lynne Berry, Courtney B. Betts, Julie Bletz, Katie Blise, Adrienne Boire, Genevieve Boland, Alexander Borowsky, Kristopher Bosse, Matthew Bott, Ed Boyden, James Brooks, Raphael Bueno, Erik A. Burlingame, Qiuyin Cai, Joshua Campbell, Wagma Caravan, Hassan Chaib, Joseph M. Chan, Young Hwan Chang, Deyali Chatterjee, Ojasvi Chaudhary, Alyce A. Chen, Bob Chen, Changya Chen, Chia-hui Chen, Feng Chen, Yu-An Chen, Milan G. Chheda, Koei Chin, Roxanne Chiu, Shih-Kai Chu, Rodrigo Chuaqui, Jaeyoung Chun, Luis Cisneros, Graham A. Colditz, Kristina Cole, Natalie Collins, Kevin Contrepois, Lisa M. Coussens, Allison L. Creason, Daniel Crichton, Christina Curtis, Tanja Davidsen, Sherri R. Davies, Ino de Bruijn, Laura Dellostritto, Angelo De Marzo, David G. DeNardo, Dinh Diep, Sharon Diskin, Xengie Doan, Julia Drewes, Stephen Dubinett, Michael Dyer, Jacklynn Egger, Jennifer Eng, Barbara Engelhardt, Graham Erwin, Laura Esserman, Alex Felmeister, Heidi S. Feiler, Ryan C. Fields, Stephen Fisher, Keith Flaherty, Jennifer Flournoy, Angelo Fortunato, Allison Frangieh, Jennifer L. Frye, Robert S. Fulton, Danielle Galipeau, Siting Gan, Jianjiong Gao, Long Gao, Peng Gao, Vianne R. Gao, Tim Geiger, Ajit George, Gad Getz, Marios Giannakis, David L. Gibbs, William E. Gillanders, Simon P. Goedegebuure, Alanna Gould, Kate Gowers, William Greenleaf, Jeremy Gresham, Jennifer L. Guerriero, Tuhin K. Guha, Alexander R. Guimaraes, David Gutman, Nir Hacohen, Sean Hanlon, Casey R. Hansen, Olivier Harismendy, Kathleen A. Harris, Aaron Hata, Akimasa Hayashi, Cody Heiser, Karla Helvie, John M. Herndon, Gilliam Hirst, Frank Hodi, Travis Hollmann, Aaron Horning, James J. Hsieh, Shannon Hughes, Won Jae Huh, Stephen Hunger, Shelley E. Hwang, Heba Ijaz, Benjamin Izar, Connor A. Jacobson, Samuel Janes, Reyka G. Jayasinghe, Lihua Jiang, Brett E. Johnson, Bruce Johnson, Tao Ju, Humam Kadara, Klaus Kaestner, Jacob Kagan, Lukas Kalinke, Robert Keith, Aziz Khan, Warren Kibbe, Albert H. Kim, Erika Kim, Junhyong Kim, Annette Kolodzie, Mateusz Kopytra, Eran Kotler, Robert Krueger, Kostyantyn Krysan, Anshul Kundaje, Uri Ladabaum, Blue B. Lake, Huy Lam, Rozelle Laquindanum, Ashley M. Laughney, Hayan Lee, Marc Lenburg, Carina Leonard, Ignaty Leshchiner, Rochelle Levy, Jerry Li, Christine G. Lian, Kian-Huat Lim, Jia-Ren Lin, Yiyun Lin, Qi Liu, Ruiyang Liu, William J.R. Longabaugh, Teri Longacre, Cynthia X. Ma, Mary Catherine Macedonia, Tyler Madison, Christopher A. Maher, Anirban Maitra, Netta Makinen, Danika Makowski, Carlo Maley, Zoltan Maliga, Diego Mallo, John Maris, Nick Markham, Jeffrey Marks, Daniel Martinez, Robert J. Mashl, Ignas Masilionais, Jennifer Mason, Joan Massagué, Pierre Massion, Marissa Mattar, Richard Mazurchuk, Linas Mazutis, Eliot T. McKinley, Joshua F. McMichael, Daniel Merrick, Matthew Meyerson, Julia R. Miessner, Gordon B. Mills, Meredith Mills, Suman B. Mondal, Motomi Mori, Yuriko Mori, Elizabeth Moses, Yael Mosse, Jeremy L. Muhlich, George F. Murphy, Nicholas E. Navin, Michel Nederlof, Reid Ness, Stephanie Nevins, Milen Nikolov, Ajit Johnson Nirmal, Garry Nolan, Edward Novikov, Brendan O’Connell, Michael Offin, Stephen T. Oh, Anastasiya Olson, Alex Ooms, Miguel Ossandon, Kouros Owzar, Swapnil Parmar, Tasleema Patel, Gary J. Patti, Itsik Pe'er, Tao Peng, Daniel Persson, Marvin Petty, Hanspeter Pfister, Kornelia Polyak, Kamyar Pourfarhangi, Sidharth V. Puram, Qi Qiu, Álvaro Quintanal-Villalonga, Arjun Raj, Marisol Ramirez-Solano, Rumana Rashid, Ashley N. Reeb, Mary Reid, Adam Resnick, Sheila M. Reynolds, Jessica L. Riesterer, Scott Rodig, Joseph T. Roland, Sonia Rosenfield, Asaf Rotem, Sudipta Roy, Charles M. Rudin, Marc D. Ryser, Maria Santi-Vicini, Kazuhito Sato, Deborah Schrag, Nikolaus Schultz, Cynthia L. Sears, Rosalie C. Sears, Subrata Sen, Triparna Sen, Alex Shalek, Jeff Sheng, Quanhu Sheng, Kooresh I. Shoghi, Martha J. Shrubsole, Yu Shyr, Alexander B. Sibley, Kiara Siex, Alan J. Simmons, Dinah S. Singer, Shamilene Sivagnanam, Michal Slyper, Artem Sokolov, Sheng-Kwei Song, Austin Southard-Smith, Avrum Spira, Janet Stein, Phillip Storm, Elizabeth Stover, Siri H. Strand, Timothy Su, Damir Sudar, Ryan Sullivan, Lea Surrey, Mario Suvà, Nadezhda V. Terekhanova, Luke Ternes, Lisa Thammavong, Guillaume Thibault, George V. Thomas, Vésteinn Thorsson, Ellen Todres, Linh Tran, Madison Tyler, Yasin Uzun, Anil Vachani, Eliezer Van Allen, Simon Vandekar, Deborah J. Veis, Sébastien Vigneau, Arastoo Vossough, Angela Waanders, Nikhil Wagle, Liang-Bo Wang, Michael C. Wendl, Robert West, Chi-yun Wu, Hao Wu, Hung-Yi Wu, Matthew A. Wyczalkowski, Yubin Xie, Xiaolu Yang, Clarence Yapp, Wenbao Yu, Yinyin Yuan, Dadong Zhang, Kun Zhang, Mianlei Zhang, Nancy Zhang, Yantian Zhang, Yanyan Zhao, Daniel Cui Zhou, Zilu Zhou, Houxiang Zhu, Qin Zhu, Xiangzhu Zhu, Yuankun Zhu, and Xiaowei Zhuang
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Cell ,Genomics ,Computational biology ,Tumor initiation ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Metastasis ,03 medical and health sciences ,Atlases as Topic ,0302 clinical medicine ,Neoplasms ,Tumor Microenvironment ,medicine ,Humans ,Precision Medicine ,030304 developmental biology ,0303 health sciences ,Atlas (topology) ,Cancer ,medicine.disease ,3. Good health ,Human tumor ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,Single-Cell Analysis ,Single point ,030217 neurology & neurosurgery - Abstract
Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
- Published
- 2020
7. Worldwide Assessment of the Mandibular First Molar Second Distal Root and Root Canal: A Cross-sectional Study with Meta-analysis
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Fábio Santiago, Luiza Berti, Adam Monroe, Yuerong Zhang, Gianluca Plotino, Carlos Boveda, Imran Cassim, Christian Nole, Antonis Chaniotis, Murilo von Zuben, Hani F. Ounsi, Hussam Alfawaz, Jorge N.R. Martins, Daniel Flynn, Marco Aurélio Versiani, Emmanuel João Nogueira Leal Silva, Hussein C. Seedat, Moataz-Bellah A.M. Alkhawas, Jojo Kottoor, Peter Parashos, Ruben Rosas Aguilar, Jose Antonio Gonzalez, Zaher Altaki, Magnús F. Ragnarsson, and Walter Vargas
- Subjects
Molar ,Male ,medicine.medical_specialty ,Cross-sectional study ,Root canal ,Dentistry ,Mandible ,Mandibular first molar ,stomatognathic system ,Statistical significance ,Medicine ,Humans ,Tooth Root ,General Dentistry ,Aged ,business.industry ,Reproducibility of Results ,Odds ratio ,Cone-Beam Computed Tomography ,Endodontics ,medicine.anatomical_structure ,Cross-Sectional Studies ,Meta-analysis ,Female ,Dental Pulp Cavity ,business - Abstract
This study assessed the prevalence of radix entomolaris and 2 canals at the distal aspect of mandibular first molars among different geographic regions by means of cone-beam computed tomographic imaging.Precalibrated observers from 23 worldwide geographic locations followed a standardized screening protocol to assess 5750 cone-beam computed tomographic images of mandibular first molars (250 per region), gathering demographic data and recording the presence of radix entomolaris and a second canal at the distal aspect of teeth. Intra- and interrater reliability tests were conducted and comparisons among groups were performed using proportions and odds ratio forest plots. The significance level was set at 5%.The results of intra- and interrater tests were above 0.79. The prevalence of radix entomolaris varied from 0.9% in Venezuela (95% confidence interval [CI], 0%-1.9%) to 22.4% in China (95% CI, 17.2%-27.6%). Regarding the proportion of a second distal canal, it ranged from 16.4% in Venezuela (95% CI, 11.8%-21.0%) to 60.0% in Egypt (95% CI, 53.9%-66.1%). The East Asia subgroup was associated with a significantly higher prevalence of an extra distolingual root, whereas the American subgroup, the American native ethnic group, and elderly patients were linked to significantly lower percentages of a second canal at the distal aspect of teeth. No significant differences were noted between male or female patients.The overall worldwide prevalence rates of radix entomolaris and a second canal at the distal aspect of the mandibular first molar were 5.6% and 36.9%, respectively. The East Asia geographic region and Asian ethnic group had a higher prevalence of a second distal root.
- Published
- 2021
8. Detection of negative allosteric effects between monoclonal antibodies by using an antigenic model-builder computer program.
- Author
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Ruben C. Aguilar, Viviana C. Blank, Lilia A. Retegui, and Leonor P. Roguin
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- 1998
- Full Text
- View/download PDF
9. Worldwide Prevalence of a Lingual Canal in Mandibular Premolars: A Multicenter Cross-sectional Study with Meta-analysis
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Yuerong Zhang, Fábio Santiago, Carlos Boveda, Luiza Berti, Christian Nole, Ruben Rosas Aguilar, Marco Aurélio Versiani, Daniel Flynn, Walter Vargas, Hussein C. Seedat, Moataz-Bellah A.M. Alkhawas, Peter Parashos, Jorge N.R. Martins, Adam Monroe, Jojo Kottoor, Gianluca Plotino, Zaher Altaki, Magnús F. Ragnarsson, Imran Cassim, Hussam Alfawaz, Jose Antonio Gonzalez, Murilo von Zuben, Emmanuel João Nogueira Leal Silva, Hani F. Ounsi, and Antonis Chaniotis
- Subjects
0301 basic medicine ,Male ,Intraclass correlation ,Cross-sectional study ,Root canal ,Dentistry ,Mandible ,03 medical and health sciences ,0302 clinical medicine ,Cohen's kappa ,stomatognathic system ,Statistical significance ,otorhinolaryngologic diseases ,medicine ,Premolar ,Prevalence ,Humans ,Multicenter Studies as Topic ,Bicuspid ,Tooth Root ,General Dentistry ,business.industry ,030206 dentistry ,Odds ratio ,Cone-Beam Computed Tomography ,Middle Aged ,030104 developmental biology ,medicine.anatomical_structure ,Cross-Sectional Studies ,Female ,sense organs ,Dental Pulp Cavity ,business ,Kappa - Abstract
The presence of multiple root canals is an important morphologic aspect of mandibular premolars. This study aimed to perform a worldwide analysis on the prevalence of a lingual canal in mandibular premolars and to evaluate its influence on patients' demographics in 23 countries using cone-beam computed tomographic images.Observers from 23 countries were instructed to evaluate cone-beam computed tomographic images of 300 first and 300 second premolars (13,800 teeth) regarding the presence of a lingual canal, canal configuration, and data related to patients' ethnicity, age, and sex following a standardized screening methodology. Intra- and interrater evaluations were performed using the Cohen kappa test and intraclass correlation coefficient. Proportion and odds ratio forest plots were calculated in order to compare groups. Statistical significance was set at 5%.Both kappa and intraclass correlation coefficient values were above 0.60, and the percentage of agreement was 94.9% (first premolar) and 97.8% (second premolar). A significant statistical difference was observed between the worldwide proportion of a lingual canal in mandibular first (23.8%; range, 12.0%-32.7%) and second (5.3%; range, 1.0%-15.3%) premolars (P.05). Asians and patients over 60 years old were associated with the lowest proportions of a lingual canal (P.05), whereas Africans and younger groups were associated with the highest proportions (P.05). The prevalence of a lingual canal in males (27.9%) was higher than females (20.0%) for the first premolar only (P.05). Males were associated with 1.533 and 1.597 higher odds of presenting a lingual root canal in the first and second premolars, respectively.The worldwide proportion of a lingual root canal was 23.6% and 5.3% for the first and second premolars, respectively. Ethnicity, geographic region, age, and sex had an influence on the outcomes.
- Published
- 2020
10. A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera
- Author
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Ruben C. Aguilar, Mike Lu, Swetha Ramadesikan, Nicholas L. Truex, Erin M. Kischuk, Andrew J. McCluskey, John Collier, Bradley L. Pentelute, Hristos Z. Kaimakliotis, Timothy L. Ratliff, Sneha Subramanian, Michael S. Santos, Deborah W. Knapp, Sherwin Jack, Bennett D. Elzey, Alexander R. Loftis, Amy E. Rabideau, Deepika Dhawan, Kayalvizhi Madhivanan, Michael O. Koch, and Daniel F. Edwards
- Subjects
Male ,Cancer Research ,Programmed cell death ,Receptor, ErbB-2 ,Anthrax toxin ,Bacterial Toxins ,Primary Cell Culture ,Antineoplastic Agents ,Apoptosis ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Dogs ,In vivo ,Cell Line, Tumor ,Medicine ,Animals ,Humans ,Antigens, Bacterial ,Bladder cancer ,Epidermal Growth Factor ,business.industry ,Immunotoxins ,medicine.disease ,In vitro ,Administration, Intravesical ,Treatment Outcome ,Oncology ,Urinary Bladder Neoplasms ,030220 oncology & carcinogenesis ,Cancer cell ,Toxicity ,Cancer research ,Female ,Drug Screening Assays, Antitumor ,business - Abstract
Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC(50) < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.
- Published
- 2019
11. Intracanal Cryotherapy Reduces Postoperative Pain in Teeth with Symptomatic Apical Periodontitis: A Randomized Multicenter Clinical Trial
- Author
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Alvaro Cruz, Philippe Sleiman, Ana Arias, Ruben Rosas Aguilar, Cesar Omar Ramos-Gregorio, Marino Vazquez-Carcaño, Jorge Ochoa, Monica Romero, and Jorge Vera
- Subjects
Adult ,Male ,medicine.medical_specialty ,Visual analogue scale ,medicine.medical_treatment ,Analgesic ,Cryotherapy ,Sodium Chloride ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Randomized controlled trial ,law ,Dental Pulp Necrosis ,medicine ,Humans ,Prospective Studies ,030212 general & internal medicine ,Therapeutic Irrigation ,General Dentistry ,Saline ,Pulp necrosis ,Periodontitis ,Pain, Postoperative ,business.industry ,030206 dentistry ,Middle Aged ,medicine.disease ,Surgery ,Clinical trial ,Anesthesia ,Female ,Dental Pulp Cavity ,business ,Periapical Periodontitis - Abstract
Introduction A prospective, multicentered, randomized clinical trial was designed to assess if controlled irrigation with cold saline could result in less incidence and intensity of postoperative pain in patients presenting with pulp necrosis and symptomatic apical periodontitis. Methods A total of 210 patients (presenting with necrotic uniradicular teeth with a diagnosis of symptomatic apical periodontitis and a preoperative visual analog scale (VAS) score higher than 7) were randomly allocated in the control or experimental group after the completion of shaping and cleaning procedures. The experimental group received a final irrigation with 20 mL sterile cold (2.5°C) saline solution delivered to the working length with a sterile, cold (2.5°C) Endovac microcannula (Kerr Endo, Orange Country, CA) for 5 minutes. The same protocol was used in the control group with room temperature saline solution. Patients were instructed to record the presence, duration and level of postoperative pain, and analgesic medication intake. A logistic regression was used to compare the incidence of postoperative pain and the need for painkillers between groups. Differences in general pain intensity between groups were analyzed using the ordinal (linear) chi-square test. Postoperative pain after 6, 24, and 72 hours (recorded in a VAS scale) and the need for analgesic medication intake between the 2 groups were assessed using the Mann-Whitney U test. Results Patients in the control group presented a significantly higher incidence of postoperative pain, intensity, and need for medication intake (P Conclusions Cryotherapy reduced the incidence of postoperative pain and the need for medication intake in patients presenting with a diagnosis of necrotic pulp and symptomatic apical periodontitis.
- Published
- 2018
12. Allosteric activators of Ocrl1: a novel therapeutic strategy against Lowe syndrome
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Swetha Ramadesikan, Kayalvizhi Madhivanan, Ruben C. Aguilar, Jennifer Lee, and Lisette Skiba
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business.industry ,Allosteric regulation ,Genetics ,Medicine ,business ,Molecular Biology ,Biochemistry ,Neuroscience ,Biotechnology ,Therapeutic strategy - Published
- 2020
13. Kidney-differentiated cells derived from Lowe Syndrome patient’s iPSCs show ciliogenesis defects and Six2 retention at the Golgi complex
- Author
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Donna M. Fekete, Ruben C. Aguilar, Wen-Chieh Hsieh, and Swetha Ramadesikan
- Subjects
0301 basic medicine ,Cellular differentiation ,Immunofluorescence ,030232 urology & nephrology ,lcsh:Medicine ,Golgi Apparatus ,Gene Expression ,Kidney ,Biochemistry ,0302 clinical medicine ,Animal Cells ,Medicine and Health Sciences ,lcsh:Science ,Induced pluripotent stem cell ,Connective Tissue Cells ,Multidisciplinary ,Secretory Pathway ,Cilium ,Stem Cells ,Cell Differentiation ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Connective Tissue ,Cell Processes ,Anatomy ,Cellular Types ,Cellular Structures and Organelles ,Reprogramming ,Research Article ,Cell type ,Mesenchyme ,Induced Pluripotent Stem Cells ,Nerve Tissue Proteins ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Ciliogenesis ,DNA-binding proteins ,medicine ,Genetics ,Humans ,Gene Regulation ,Cell Lineage ,Cilia ,Immunoassays ,Homeodomain Proteins ,lcsh:R ,Biology and Life Sciences ,Proteins ,Kidneys ,Renal System ,Cell Biology ,Fibroblasts ,Regulatory Proteins ,030104 developmental biology ,Biological Tissue ,Oculocerebrorenal Syndrome ,Cell culture ,Immunologic Techniques ,lcsh:Q ,Developmental Biology ,Transcription Factors - Abstract
Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the OCRL1 gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient's cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs) from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, OCRL1 KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2's role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.
- Published
- 2018
14. Worldwide Prevalence of Mandibular Second Molar C-Shaped Morphologies Evaluated by Cone-Beam Computed Tomography
- Author
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Ruben Rosas Aguilar, Murilo von Zuben, Daniel Flynn, Miguel Seruca Marques, Jojo Kottoor, Luiza Berti, Imran Cassim, Yongchun Gu, António Ginjeira, Adam Monroe, Jose Antonio Gonzalez, and Jorge N.R. Martins
- Subjects
Molar ,Adult ,Male ,Cone beam computed tomography ,Independent group ,Root canal ,Dentistry ,Computed tomography ,Mandible ,Global Health ,Mandibular second molar ,03 medical and health sciences ,0302 clinical medicine ,C shaped ,medicine ,Prevalence ,Radiography, Dental ,Humans ,General Dentistry ,Orthodontics ,medicine.diagnostic_test ,business.industry ,030206 dentistry ,Intra-rater reliability ,Cone-Beam Computed Tomography ,medicine.anatomical_structure ,Cross-Sectional Studies ,030220 oncology & carcinogenesis ,Female ,business - Abstract
Introduction The aim of this study was to evaluate and compare the C-shaped mandibular second molar prevalence in different regions around the world with the aid of cone-beam computed tomography technology. Methods Nine field observers from 9 different geographic regions were calibrated. A total of 400 samples were collected in each region. The prevalence of C-shaped anatomy was calculated. The number of roots and the configuration of the C-shaped canals at 3 different axial levels were also evaluated. The z -test was used to analyze the difference between the means of each independent group. Intrarater reliability was also tested. Results A total of 3600 teeth from 2735 patients were included in this research; 499 teeth presented C-shaped root canal configuration, representing a global prevalence of 13.9%. China had a prevalence of 44.0%, which was significantly higher than any other region. The C-shape prevalence in women was 16.5%, which was significantly higher than the 10.4% prevalence found in men. No difference between sides (37 or 47) was evident in the global sample. Conclusions Cone-beam computed tomography is a valuable tool to evaluate the C-shaped root canal configuration in vivo . In the present study, China presented the highest prevalence of C-shaped mandibular second molars when compared with other regions. Women exhibited a higher prevalence than men.
- Published
- 2017
15. Ciliopathies: The Trafficking Connection
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Ruben C. Aguilar and Kayalvizhi Madhivanan
- Subjects
Polycystic Kidney Diseases ,Cilium ,Cell Biology ,Biology ,Biochemistry ,Phenotype ,Ciliopathies ,Article ,Transport protein ,Cell biology ,Protein Transport ,Structural Biology ,Organelle ,Genetics ,Animals ,Humans ,Ciliary Motility Disorders ,Cilia ,Molecular Biology ,Ciliary base ,Secretory pathway - Abstract
The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle-dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multiorgan diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies appears to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e. affecting cilia assembly) will lead to more severe symptoms, whereas those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology.
- Published
- 2014
16. Worldwide Analyses of Maxillary First Molar Second Mesiobuccal Prevalence: A Multicenter Cone-beam Computed Tomographic Study
- Author
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Ruben Rosas Aguilar, Zaher Altaki, Hani F. Ounsi, Fábio Santiago, Yuerong Zhang, Magnús F. Ragnarsson, Luiza Berti, Daniel Flynn, Adam Monroe, Gianmarco Bellardini, Carlos Boveda, Jojo Kottoor, Jose Antonio Gonzalez, Gianluca Plotino, Jorge N.R. Martins, Walter Vargas, Yongchun Gu, Antonis Chaniotis, Miguel Seruca Marques, António Ginjeira, Murilo von Zuben, Hussein C. Seedat, Moataz-Bellah A.M. Alkhawas, and Peter Parashos
- Subjects
Adult ,Male ,0301 basic medicine ,Molar ,Patient demographics ,Root canal ,Dentistry ,Global Health ,Computed tomographic ,Young Adult ,03 medical and health sciences ,Sex Factors ,0302 clinical medicine ,Maxilla ,Prevalence ,Maxillary first molar ,Humans ,Medicine ,Tooth Root ,General Dentistry ,business.industry ,Age Factors ,Anatomic Variation ,030206 dentistry ,Cone-Beam Computed Tomography ,Middle Aged ,030104 developmental biology ,medicine.anatomical_structure ,Mesiobuccal root ,Sample size determination ,Female ,Dental Pulp Cavity ,business - Abstract
Maxillary first molar second mesiobuccal (MB2) root canal prevalence may change among different populations. The aim of this study was to analyze the worldwide prevalence of the MB2 root canal and understand its possible relation with sex, age, side, and root configuration using in vivo cone-beam computed tomographic (CBCT) assessment.Observers from 21 regions were calibrated to achieve a similar CBCT assessment methodology and instructed to collect data from 250 maxillary first molars in previously existing examinations. Intra- and interrater reliability tests were performed. The sample size included 5250 molars and was defined by way of a preliminary trial. Data collected included MB2 presence, sex, age, side, number of roots per tooth, and mesiobuccal root configuration. The z test for proportions in independent groups was used to analyze the differences among subgroups. P .05 was considered significant.The worldwide CBCT-assessed MB2 prevalence was 73.8%, ranging from 48.0% in Venezuela to 97.6% in Belgium. The prevalence in males and females was 76.3% and 71.8%, respectively (P .05). Significantly higher MB2 proportions were found in younger patients and 3-rooted molar configurations. The group intraclass correlation coefficient and the percentage of agreement for the MB2 presence were 0.95 and 0.91, respectively. The intrarater Cohen kappa value was above 0.61 for all observers.MB2 prevalence in the analyzed regions varied widely. The differences may be associated with specificities within each region but also patient demographics. Males, younger patients, and 3-rooted configurations were associated with higher MB2 proportions.
- Published
- 2018
17. Adaptor Proteins: Inter-Organelle Traffic Controllers
- Author
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Kayalvizhi Madhivanan, Wen-Chieh Hsieh, and Ruben C. Aguilar
- Subjects
Epsin ,biology ,Signal transducing adaptor protein ,macromolecular substances ,medicine.disease_cause ,Clathrin ,Cell biology ,Organelle ,Protein targeting ,biology.protein ,medicine ,Compartment (development) ,Ap180 ,Function (biology) - Abstract
The complex biochemical activities that take place within the cell require a precise spatiotemporal organization of its components. This dynamic intracellular order is generated and maintained by a transport system based on membrane-bound carriers that pinch off from a donor and deliver cargoes into a target compartment. The crucial task of selecting the cargoes to be transported from one compartment to another is the responsibility of the protein machinery that coats the cytosolic side of the carriers until they bud off. These coats contain specialized components known as ‘adaptors’ that interpret cargo destination information and include them or not into the nascent transport carrier. This article reviews several families of adaptors and summarizes their function, specificity, and cellular/organism relevance.
- Published
- 2016
18. The Lowe syndrome protein OCRL1 is involved in primary cilia assembly
- Author
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I Barinaga-Rementeria Ramirez, Kayalvizhi Madhivanan, Debarati Mukherjee, Philip L. Beales, Claudia B. Hanna, Brian G. Coon, Martin Lowe, V Hernandez, and Ruben C. Aguilar
- Subjects
Embryo, Nonmammalian ,Recombinant Fusion Proteins ,Oculocerebrorenal syndrome ,Endosomes ,Disease ,Biology ,Bioinformatics ,Ciliopathies ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,medicine ,Animals ,Humans ,Cilia ,Antigens ,RNA, Small Interfering ,Molecular Biology ,Zebrafish ,Cells, Cultured ,Genetics (clinical) ,030304 developmental biology ,0303 health sciences ,Cilium ,Interleukin-2 Receptor alpha Subunit ,Cell Biology ,General Medicine ,medicine.disease ,biology.organism_classification ,Phenotype ,Human genetics ,Phosphoric Monoester Hydrolases ,3. Good health ,Disease Models, Animal ,Protein Transport ,Ciliopathy ,Oculocerebrorenal Syndrome ,Poster Presentation ,Congenital cataracts ,OCRL ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.
- Published
- 2012
19. Granulocyte colony-stimulating factor (G-CSF) upregulates β1 integrin and increases migration of human trophoblast Swan 71 cells via PI3K and MAPK activation
- Author
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Leonor P. Roguin, Ruben C. Aguilar, Julieta Marino, Graciela Cremaschi, Maria Florencia Cayrol, Verónica Alejandra Furmento, and Viviana C. Blank
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,medicine.medical_specialty ,MAP Kinase Signaling System ,Motility ,Biology ,Células Trofoblásticas ,Article ,purl.org/becyt/ford/1 [https] ,QUIMICA BIOLOGICA ,03 medical and health sciences ,chemistry.chemical_compound ,Phosphatidylinositol 3-Kinases ,Cell Movement ,BIOLOGIA ,Internal medicine ,Cell Line, Tumor ,Granulocyte Colony-Stimulating Factor ,medicine ,Humans ,PLACENTA ,purl.org/becyt/ford/1.6 [https] ,PI3K/AKT/mTOR pathway ,Integrin beta1 ,Migración ,Trophoblast ,Cell migration ,EMBRION HUMANO ,Cell Biology ,Gcsf ,Actin cytoskeleton ,Cell biology ,Trophoblasts ,Up-Regulation ,Vascular endothelial growth factor ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Tissue Array Analysis ,Integrina ,Signal transduction ,Mitogen-Activated Protein Kinases - Abstract
Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of β1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of β1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates β1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development. Fil: Furmento, Verónica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Marino, Veronica Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Blank, Viviana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Cayrol, Maria Florencia. Pontificia Universidad Católica Argentina ; Argentina Fil: Cremaschi, Graciela Alicia. Pontificia Universidad Católica Argentina ; Argentina Fil: Aguilar, Ruben Claudio. Purdue University; Estados Unidos Fil: Roguin, Leonor Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina
- Published
- 2015
20. The Yeast Epsin Ent1 Is Recruited to Membranes through Multiple Independent Interactions
- Author
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Beverly Wendland, Hadiya A. Watson, and Ruben C. Aguilar
- Subjects
Models, Molecular ,Fungal protein ,Saccharomyces cerevisiae Proteins ,Epsin ,Ubiquitin ,Membrane lipids ,Cell Membrane ,Endocytic cycle ,Vesicular Transport Proteins ,Biological membrane ,Cell Biology ,Biology ,Phosphatidylinositols ,Biochemistry ,Endocytosis ,Cell biology ,Fungal Proteins ,biology.protein ,Carrier Proteins ,ENTH domain ,Molecular Biology - Abstract
In addition to its well known role in targeting proteins for proteasomal degradation, ubiquitin (Ub) is also involved in promoting internalization of cell surface proteins into the endocytic pathway. Moreover, putative Ub interaction motifs (UIMs) as well as Ub-associated (UBA) domains have been identified in key yeast endocytic proteins (the epsins Ent1 and Ent2, and the Eps15 homolog Ede1). In this study, we characterized the interaction of Ub with the Ede1 UBA domain and with the UIMs of Ent1. Our data suggest that the UIMs and the UBA are involved in binding these proteins to biological membranes. We also show that the Ent1 ENTH domain binds to phosphoinositides in vitro and that Ent1 NPF motifs interact with the EH domain-containing proteins Ede1 and Pan1. Our findings indicate that the ENTH domain interaction with membrane lipids cooperates with the binding of membrane-associated Ub moieties. These events may in turn favor the occurrence of other interactions, for instance EH-NPF recognition, thus stabilizing networks of low affinity binding partners at endocytic sites.
- Published
- 2003
21. Functional and physical interactions of the adaptor protein complex AP-4 with ADP-ribosylation factors (ARFs)
- Author
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Juan S. Bonifacino, Markus Boehm, and Ruben C. Aguilar
- Subjects
ADP ribosylation factor ,GTP' ,Immunoblotting ,Golgi Apparatus ,Plasma protein binding ,Biology ,Transfection ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,Protein structure ,Two-Hybrid System Techniques ,Humans ,Molecular Biology ,Integral membrane protein ,Binding Sites ,Brefeldin A ,General Immunology and Microbiology ,ADP-Ribosylation Factors ,General Neuroscience ,Cell Membrane ,Membrane Proteins ,Signal transducing adaptor protein ,Precipitin Tests ,Protein Structure, Tertiary ,Cell biology ,Vesicular transport protein ,Adaptor Proteins, Vesicular Transport ,Microscopy, Fluorescence ,Membrane protein ,Mutation ,Mutagenesis, Site-Directed ,Carrier Proteins ,HeLa Cells ,Protein Binding ,Signal Transduction - Abstract
AP-4 is a member of the family of heterotetrameric adaptor protein (AP) complexes that mediate the sorting of integral membrane proteins in post-Golgi compartments. This complex consists of four subunits (epsilon, beta4, mu4 and sigma4) and localizes to the cytoplasmic face of the trans-Golgi network (TGN). Here, we show that the recruitment of endogenous AP-4 to the TGN in vivo is regulated by the small GTP-binding protein ARF1. In addition, we demonstrate a direct interaction of the epsilon and mu4 subunits of AP-4 with ARF1. epsilon binds only to ARF1-GTP and requires residues in the switch I and switch II regions of ARF1. In contrast, mu4 binds equally well to the GTP- and GDP-bound forms of ARF1 and is less dependent on switch I and switch II residues. These observations establish AP-4 as an ARF1 effector and suggest a novel mode of interaction between ARF1 and an AP complex involving both constitutive and regulated interactions.
- Published
- 2001
22. Stonin 2
- Author
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Ruben C. Aguilar, Jose A. Martina, Juan S. Bonifacino, and Cecilia J. Bonangelino
- Subjects
Synaptotagmins ,biology ,Endosome ,Endocytic cycle ,biology.protein ,Signal transducing adaptor protein ,Adaptor Signaling Protein ,Cell Biology ,Plasma protein binding ,Clathrin ,Adaptor Protein Complex alpha Subunits ,Cell biology - Abstract
Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Here, we report the identification of two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery. The human stonins are homologous to the Drosophila melanogaster stoned B protein and exhibit a modular structure consisting of an NH2-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the μ subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP–stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery.
- Published
- 2001
23. Signal-binding Specificity of the μ4 Subunit of the Adaptor Protein Complex AP-4
- Author
-
Hiroshi Ohno, Inna Gorshkova, Markus Boehm, Ruben C. Aguilar, Takashi Saito, Kazuhiro Tomita, Juan S. Bonifacino, and Robert J. Crouch
- Subjects
Recombinant Fusion Proteins ,Protein subunit ,Molecular Sequence Data ,Nerve Tissue Proteins ,Saccharomyces cerevisiae ,Biology ,Transfection ,Biochemistry ,Antigens, CD ,Peptide Library ,Aspartic acid ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Tyrosine ,Peptide library ,Molecular Biology ,Integral membrane protein ,Binding selectivity ,Binding Sites ,Membrane Glycoproteins ,Lysosome-Associated Membrane Glycoproteins ,Signal transducing adaptor protein ,Cell Biology ,Phosphoproteins ,Recombinant Proteins ,Adaptor Proteins, Vesicular Transport ,Protein Subunits ,Amino Acid Substitution ,Cytoplasm ,Monomeric Clathrin Assembly Proteins ,Mutagenesis, Site-Directed ,Lysosomes ,HeLa Cells ,Signal Transduction - Abstract
The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.
- Published
- 2001
24. Ggas
- Author
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Juan S. Bonifacino, Chris Mullins, Esteban C. Dell'Angelica, Jose D. Vargas, Lisa M. Hartnell, Ruben C. Aguilar, and Rosa Puertollano
- Subjects
Cytoplasm ,ADP ribosylation factor ,Recombinant Fusion Proteins ,Immunoelectron microscopy ,Genes, Fungal ,Molecular Sequence Data ,Cathepsin A ,Fluorescent Antibody Technique ,Golgi Apparatus ,Coated vesicle ,Carboxypeptidases ,Saccharomyces cerevisiae ,Biology ,adaptins ,symbols.namesake ,chemistry.chemical_compound ,Humans ,Cloning, Molecular ,VHS ,Microscopy, Immunoelectron ,Adaptor Protein Complex gamma Subunits ,coats ,Brefeldin A ,ADP-Ribosylation Factors ,trans-Golgi network ,ADP-ribosylation factor binding ,Membrane Proteins ,Proteins ,Biological Transport ,Coated Pits, Cell-Membrane ,Cell Biology ,COPI ,Golgi apparatus ,Protein Structure, Tertiary ,Transport protein ,Cell biology ,Molecular Weight ,Adaptor Proteins, Vesicular Transport ,chemistry ,Mutation ,Vacuoles ,symbols ,Original Article ,ADP-Ribosylation Factor 1 ,Carrier Proteins ,HeLa Cells ,Protein Binding - Abstract
Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of γ-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN.
- Published
- 2000
25. Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXphi targeting signals
- Author
-
Nancy R. Gough, Olga Martinez-Augustin, Ruben C. Aguilar, Mark E. Zweifel, Juan S. Bonifacino, and Douglas M. Fambrough
- Subjects
Signal peptide ,Endosome ,Recombinant Fusion Proteins ,media_common.quotation_subject ,Mutant ,Biology ,Transfection ,Cell membrane ,Mice ,L Cells ,Antigens, CD ,Genes, Reporter ,Lysosome ,medicine ,Animals ,Lysosome-associated membrane glycoprotein ,Amino Acid Sequence ,Cycloheximide ,Internalization ,media_common ,Membrane Glycoproteins ,Microscopy, Confocal ,Cell Membrane ,Lysosome-Associated Membrane Glycoproteins ,Cell Biology ,Peptide Fragments ,Transmembrane protein ,Cell biology ,Butyrates ,medicine.anatomical_structure ,Gene Expression Regulation ,Binding Sites, Antibody ,Lysosomes - Abstract
A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.
- Published
- 1999
26. μ1B, a novel adaptor medium chain expressed in polarized epithelial cells1
- Author
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Hiroshi Ohno, Heike Foelsch, Takuya Tomemori, Takashi Saito, Takuji Shirasawa, Yasushi Okazaki, Fubito Nakatsu, Juan S. Bonifacino, Ruben C. Aguilar, and Ira Mellman
- Subjects
Clathrin adaptor complex ,Biophysics ,Signal transducing adaptor protein ,Cell Biology ,Basolateral plasma membrane ,Biology ,medicine.disease_cause ,Endocytosis ,Biochemistry ,Molecular biology ,Cell biology ,Structural Biology ,Basolateral Sorting Signal ,Protein targeting ,Genetics ,medicine ,Molecular Biology ,Peptide sequence ,Integral membrane protein - Abstract
The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins. Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting. Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting. Here, we report the identification of a novel member of the adaptor medium chain family, named μ1B, which is closely related to the previously described μ1A (79% amino acid sequence identity). Northern blotting and in situ hybridization analyses reveal the specific expression of μ1B mRNA in a subset of polarized epithelial and exocrine cells. Yeast two-hybrid analyses show that μ1B is capable of interacting with generic tyrosine-based sorting signals. These observations suggest that μ1B may be involved in protein sorting events specific to polarized cells.
- Published
- 1999
27. Functional Domain Mapping of the Clathrin-associated Adaptor Medium Chains ॖ1 and ॖ2
- Author
-
Ruben C. Aguilar, Katherine W. Roche, Hiroshi Ohno, and Juan S. Bonifacino
- Subjects
Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Adaptor Protein Complex 3 ,Macromolecular Substances ,Cloning, Organism ,Recombinant Fusion Proteins ,Adaptor Protein Complex 1 ,Molecular Sequence Data ,Adaptor Protein Complex 2 ,Nerve Tissue Proteins ,Saccharomyces cerevisiae ,Biochemistry ,Clathrin ,Antibodies ,Fungal Proteins ,Two-Hybrid System Techniques ,Molecule ,Amino Acid Sequence ,Tyrosine ,Molecular Biology ,Integral membrane protein ,Sequence Deletion ,biology ,Sorting ,Proteolytic enzymes ,Cell Biology ,Phosphoproteins ,beta-Galactosidase ,Yeast ,Adaptor Protein Complex mu Subunits ,DNA-Binding Proteins ,Adaptor Proteins, Vesicular Transport ,Kinetics ,Cytosol ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,biology.protein ,Biophysics ,Protein Multimerization ,Transcription Factors - Abstract
The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins. The medium chains of these complexes, mu1 and mu2, have been implicated in two types of interaction: assembly with the beta1 and beta2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals. In this study, we report the results of a structure-function analysis of the mu1 and mu2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions. Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that mu1 and mu2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of mu1 and mu2) being involved in assembly with the beta chains and the carboxyl-terminal two-thirds (residues 147-423 of mu1 and 164-435 of mu2) binding tyrosine-based sorting signals. These observations support a model in which the amino-terminal one-third of mu2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.
- Published
- 1997
28. Monoclonal antibodies to human growth hormone modulate its biological properties
- Author
-
Ruben C. Aguilar, Leonor P. Roguin, and Lilia A. Retegui
- Subjects
endocrine system ,medicine.drug_class ,Immunology ,In Vitro Techniques ,Biology ,Monoclonal antibody ,Epitope ,Antigen-Antibody Reactions ,Cell membrane ,Human placental lactogen ,Allosteric Regulation ,medicine ,Humans ,Receptor ,Molecular Biology ,Cells, Cultured ,Cell growth ,Cell Membrane ,Antibodies, Monoclonal ,Biological activity ,Receptors, Somatotropin ,Prolactin ,medicine.anatomical_structure ,Epitope mapping ,Biochemistry ,Growth Hormone ,Cell Division ,Epitope Mapping ,hormones, hormone substitutes, and hormone antagonists - Abstract
Previous results indicated that monoclonal antibodies (mAbs) termed mAb AE5, mAb AC8 and mAb F11, recognizing the human growth hormone (hGH) region left exposed after binding to lactogenic, somatogenic and hGH-specific receptors, produce allosteric changes in the hormone which modify its binding properties. To study whether these mAbs could also influence hGH biological activity, experiments were carried out with Nb2 cells, a rat lymphoma cell line which proliferates in the presence of lactogenic hormones. Experiments involving previous binding of the hormone to receptors before adding 125I-mAbs indicated that the hGH domain defined by overlapped epitopes AE5, AC8 and F11 is uncovered in hGH when it is bound to the cell membranes. To reveal any alteration in the hGH molecule induced by the mAbs, preformed 125I-mAb:hGH complexes were added to the cell membranes. Data showed that 125I-mAb AE5:hGH complexes bound better to the receptors than free hormone. On the contrary, hGH previously bound to 125I-mAb AC8 or 125I-mAb F11 was poorly recognized by Nb2 receptors. Furthermore, both mAbs AC8 and F11 strongly inhibited 125I-hGH binding to Nb2 cell membranes and hGH-induced Nb2 cell proliferation whereas mAb AE5 enhanced both hormone binding and hGH mitogenic effect. Additionally, since mAb AC8 is directed towards an epitope shared by hGH and human placental lactogen (hPL), it was also shown that this mAb could impair hPL biological activity even though it recognizes the hPL region left exposed in hPL:Nb2 cell receptor complexes. Data presented in this work suggest that mAbs directed to the hGH or hPL regions unmasked after binding to Nb2 cell receptors produce allosteric alterations in the binding properties of these hormones leading to either enhancement or decrease of their biological activities.
- Published
- 1995
29. MAPAG: a computer program to construct 2- and 3-dimensional antigenic maps
- Author
-
Leonor P. Roguin, Ruben C. Aguilar, and Lilia A. Retegui
- Subjects
Computer program ,Group method of data handling ,Antibodies, Monoclonal ,Reproducibility of Results ,Medicine (miscellaneous) ,Experimental data ,Bayes Theorem ,Construct (python library) ,Luteinizing Hormone ,Peptide Mapping ,Epitope ,Rats ,Epitopes ,User-Computer Interface ,Microcomputers ,Simple (abstract algebra) ,Growth Hormone ,Animals ,Logical matrix ,Graphics ,Algorithm ,Algorithms ,Software ,Mathematics - Abstract
The contact area between an antibody (Ab) and the antigen (Ag) is called antigenic determinant or epitope. The first step in the characterization of an Ag by using monoclonal antibodies (MAb) is to map the relative distribution of the corresponding epitopes on the Ag surface. The computer program MAPAG has been devised to automatically construct antigenic maps. MAPAG is fed with a binary matrix of experimental data indicating the ability of paired MAb to bind or not simultaneously to the Ag. The program is interactive menu-driven and allows the user an easy data handling. MAPAG utilizes iterative processes to construct and to adjust the final map, which is graphically shown as a 2- or a 3-dimensional model. Additionally, the antigenic map obtained can be optionally modified by the user or readjusted by the program. The suitability of MAPAG was illustrated by running experimental data from literature and comparing antigenic maps constructed by the program with those elaborated by the investigators without the assistance of a computer. Furthermore, since some MAb could present negative allosteric effects leading to misinterpretation of data, MAPAG has been provided with an approximate reasoning module to solve such anomalous situations. Results indicated that the program can be successfully employed as a simple, fast and reliable antigenic model-builder.
- Published
- 1994
30. Microfluidic based contactless dielectrophoretic device: Modeling and analysis
- Author
-
Ruben C. Aguilar, Michael Gaitan, Nathalia Peixoto, Saugandhika Minnikanti, Darwin R. Reyes, and Joseph J. Pancrazio
- Subjects
Electrophoresis ,Microchannel ,Materials science ,Microfluidics ,Electrode ,Nanotechnology ,Substrate (printing) ,Multielectrode array ,Dielectrophoresis ,Microfluidic Analytical Techniques ,Models, Theoretical ,Finite element method ,Microfabrication - Abstract
While there have been many attempts at patterning cells onto substrates, a reliable method for trapping cell clusters and forming cell arrays in a predefined geometry remains to be demonstrated. We intend to develop a multielectrode array platform to initially trap cells via dielectrophoresis (DEP) and to later measure their electrical activity. As a first step toward that objective, here we present an interdigitated microfabricated comb structure. We designed an optimal insulation layer via finite element modeling for maximum dielectrophoretic field strength in solution and minimal cell damage. The microfabricated structure was combined with a microfluidic channel to vertically constrain cell position. With the objective of capturing cells onto the substrate, we here show that there is an optimal thickness of dielectric which limits electrolysis in solution and still allows for sufficient dielectrophoretic force on the cells to pull them onto the surface.
- Published
- 2010
31. Identification of somatogenic binding sitess in liver microsomes from normal mice and transgenic mice expressing human growth hormone gene
- Author
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Andrzej Bartke, R.S. Calandra, Juan M. Dellacha, D. Turyn, H. N. Fernandez, and Ruben C. Aguilar
- Subjects
Male ,Genetically modified mouse ,medicine.medical_specialty ,Receptors, Prolactin ,Ratón ,Gene Expression ,Mice, Transgenic ,Peptide hormone ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Internal medicine ,medicine ,Animals ,General Pharmacology, Toxicology and Pharmaceutics ,Binding site ,Liver microsomes ,Gene ,biology ,Receptors, Somatotropin ,General Medicine ,biology.organism_classification ,Kinetics ,Endocrinology ,Microsoma ,Growth Hormone ,Microsomes, Liver ,Microsome ,Autoradiography ,Electrophoresis, Polyacrylamide Gel ,Female ,hormones, hormone substitutes, and hormone antagonists - Abstract
Somatogenic binding sites were detected and characterized in microsomal preparations from livers of normal mice and mice expressing metallothionein-l/hGH (mMT/hGH) hybrid gene, using 125 l-labelled bovine or human GH, or a photoreactive derivative of hGH ( 125 l-AP-hGH1). Specific binding of 125 l-bGH was detected in liver microsomes from both normal and transgenic mice with an apparent Kd of 2 nM. 125 l-hGH was partially displaced by bGH. 125 l-AP-hGH1 was covalently bound to the microsomal preparations, and bGH prevented the formation of the 130 kDa species with no appreciable effect on 63 kDa and 70 kDa lactogenic complexes.
- Published
- 1992
32. Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins
- Author
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Lan Yu Yeh, Beverly Wendland, Ariel Hsu, Arne Schön, Sean Kim, William K. McCormick, Ernesto Freire, Hadiya A. Watson, Ruben C. Aguilar, Silvia Andrea Longhi, and Jonathan D. Shaw
- Subjects
Models, Molecular ,Scaffold protein ,Saccharomyces cerevisiae Proteins ,Epsin ,GTPase-activating protein ,ENDOCYTOSIS ,Genes, Fungal ,Endocytic cycle ,Vesicular Transport Proteins ,Saccharomyces cerevisiae ,CDC42 ,Clathrin ,ACTIN ,Ciencias Biológicas ,Cell polarity ,ENTH domain ,Adaptor Proteins, Signal Transducing ,cdc42 GTP-Binding Protein, Saccharomyces cerevisiae ,Multidisciplinary ,biology ,Cell Polarity ,Bioquímica y Biología Molecular ,Endocytosis ,humanities ,Protein Structure, Tertiary ,Cell biology ,Adaptor Proteins, Vesicular Transport ,Phenotype ,Mutation ,Commentary ,biology.protein ,Carrier Proteins ,CIENCIAS NATURALES Y EXACTAS ,POLARITY - Abstract
Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Δent2Δ cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity. Fil: Aguilar, Ruben Claudio. Purdue University; Estados Unidos. University Johns Hopkins; Estados Unidos Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Shaw, Jonathan D.. University Johns Hopkins; Estados Unidos Fil: Yeh, Lan Yu. National Institutes of Health; Estados Unidos Fil: Kim, Sean. University Johns Hopkins; Estados Unidos Fil: Schön, Arne. University Johns Hopkins; Estados Unidos Fil: Freire, Ernesto. National Institutes of Health; Estados Unidos Fil: Hsu, Ariel. University Johns Hopkins; Estados Unidos Fil: McCormick, William K.. University Johns Hopkins; Estados Unidos Fil: Watson, Hadiya A.. University Johns Hopkins; Estados Unidos. National Instituto Of Child Health & Human Development; Estados Unidos Fil: Wendland, Beverly. University Johns Hopkins; Estados Unidos
- Published
- 2006
33. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor
- Author
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Cecilia N. Arighi, Ruben C. Aguilar, Carol Renfrew Haft, Lisa M. Hartnell, and Juan S. Bonifacino
- Subjects
SNX27 ,Retromer ,Endosome ,Hydrolases ,Vesicular Transport Proteins ,Down-Regulation ,Endosomes ,Biology ,Receptor, IGF Type 2 ,Article ,VPS35 ,Mice ,Animals ,Humans ,Transport Vesicles ,VPS26A ,Cell Biology ,Cell biology ,Cell Compartmentation ,Protein Structure, Tertiary ,lysosomal enzymes ,multivesicular bodies ,sorting nexins ,yeast vacuole ,clathrin adaptors ,Retromer complex ,Sorting nexin ,Microscopy, Electron ,Protein Transport ,VPS29 ,RNA Interference ,Carrier Proteins ,Lysosomes ,HeLa Cells ,trans-Golgi Network - Abstract
The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular–vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.
- Published
- 2004
34. Ubiquitin: not just for proteasomes anymore
- Author
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Ruben C. Aguilar and Beverly Wendland
- Subjects
Proteasome Endopeptidase Complex ,Proteolysis ,Active Transport, Cell Nucleus ,Receptors, Cell Surface ,Ubiquitin-conjugating enzyme ,Deubiquitinating enzyme ,Ubiquitin ,Multienzyme Complexes ,medicine ,Animals ,Humans ,Cell division control protein 4 ,biology ,medicine.diagnostic_test ,Cell Biology ,Endocytosis ,Cell biology ,Ubiquitin ligase ,Cysteine Endopeptidases ,Protein Transport ,Eukaryotic Cells ,Proteasome ,Biochemistry ,biology.protein ,Target protein ,Peptide Hydrolases - Abstract
Ubiquitin is a small protein that can be covalently linked to itself or other proteins, either as single ubiquitin molecules or as chains of polyubiquitin. Addition of ubiquitin to a target protein requires a series of enzymatic activities (by ubiquitin-activating, -conjugating and -ligating enzymes). The first function attributed to ubiquitin was the covalent modification of misfolded cytoplasmic proteins, thereby directing proteasome-dependent proteolysis. More recently, additional functions have been ascribed to ubiquitin and ubiquitin-related proteins. Ubiquitin directs specific proteins through the endocytic pathway by modifying cargo proteins, and possibly also components of the cytoplasmic protein trafficking machinery.
- Published
- 2003
35. Sorting of mannose 6-phosphate receptors mediated by the GGAs
- Author
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Robert J. Crouch, Ruben C. Aguilar, Rosa Puertollano, Juan S. Bonifacino, and Inna Gorshkova
- Subjects
Recombinant Fusion Proteins ,Mutant ,Amino Acid Motifs ,Molecular Sequence Data ,Mannose ,Mannose 6-phosphate ,Biology ,Protein Sorting Signals ,Receptor, IGF Type 2 ,Cell Line ,chemistry.chemical_compound ,symbols.namesake ,Dogs ,Lysosome ,Cations ,Two-Hybrid System Techniques ,Yeasts ,medicine ,GGA1 ,Animals ,Humans ,Amino Acid Sequence ,Receptor ,Transport Vesicles ,Multidisciplinary ,ADP-Ribosylation Factors ,Proteins ,Dipeptides ,Golgi apparatus ,Clathrin ,Protein Structure, Tertiary ,Cytosol ,Adaptor Proteins, Vesicular Transport ,Protein Transport ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Microscopy, Fluorescence ,COS Cells ,Mutation ,symbols ,Carrier Proteins ,trans-Golgi Network - Abstract
The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, γ-ear–containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.
- Published
- 2001
36. Molecular characterization of the protein encoded by the Hermansky-Pudlak syndrome type 1 gene
- Author
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William A. Gahl, Ruben C. Aguilar, Esteban C. Dell'Angelica, Senator Hazelwood, Nathan E. Wolins, and Juan S. Bonifacino
- Subjects
Vesicle-associated membrane protein 8 ,Transcription, Genetic ,Protein subunit ,Mutant ,Biology ,Biochemistry ,Cell Line ,Gene product ,Mice ,Cytosol ,Gene Duplication ,Animals ,Humans ,Molecular Biology ,Integral membrane protein ,Cells, Cultured ,Skin ,B-Lymphocytes ,CD63 ,Genetic Diseases, Inborn ,Membrane Proteins ,Cell Biology ,Intracellular Membranes ,Syndrome ,Molecular biology ,eye diseases ,Cell biology ,Membrane protein ,Lysosomes ,HeLa Cells - Abstract
Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of approximately 80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was approximately 6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the beta3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1 gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex.
- Published
- 2000
37. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor
- Author
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Juan S. Bonifacino, Ruben C. Aguilar, Vorasuk Shotelersuk, Esteban C. Dell'Angelica, and William A. Gahl
- Subjects
Protein subunit ,Mutant ,DNA Mutational Analysis ,Molecular Sequence Data ,HPS5 ,Nerve Tissue Proteins ,Platelet Membrane Glycoproteins ,Biology ,medicine.disease_cause ,AP3B1 ,Antigens, CD ,Organelle ,Protein targeting ,medicine ,Humans ,RNA, Messenger ,Molecular Biology ,Genetics ,Melanosomes ,Membrane Glycoproteins ,Tetraspanin 30 ,Lysosome-Associated Membrane Glycoproteins ,Membrane Proteins ,Proteins ,Cell Biology ,Fibroblasts ,medicine.disease ,Flow Cytometry ,Phosphoproteins ,Molecular biology ,eye diseases ,Adaptor Proteins, Vesicular Transport ,Microscopy, Fluorescence ,Albinism, Oculocutaneous ,Monomeric Clathrin Assembly Proteins ,Hermanski-Pudlak Syndrome ,Hermansky–Pudlak syndrome ,Protein Processing, Post-Translational - Abstract
Summary a component of an organellar coat involved in protein sorting. Hermansky-Pudlaksyndrome(HPS)isageneticdisor- Our studies concern the Hermansky-Pudlak syn-der characterized by defective lysosome-related or- drome (HPS), an autosomal recessive disorder charac- ganelles. Here, we report the identification of two HPS terizedby oculocutaneousalbinismand plateletstorage patients with mutations in the b 3A subunit of the het- pool deficiency (Hermansky and Pudlak, 1959). These erotetrameric AP-3 complex. The patients’ fibroblasts abnormalities arise from defects in specialized cyto- exhibit drastically reduced levels of AP-3 due to en- plasmic organelles, namely melanosomes and platelet- hanced degradation of mutant b 3A. The AP-3 de- dense granules. Over time, some HPS patients develop ficiency results in increased surface expression of pulmonary fibrosis and granulomatous colitis, presum- the lysosomal membrane proteins CD63, lamp-1, and ably due to accumulation of undegraded materials in
- Published
- 1999
38. Endocytosis of membrane receptors: Two pathways are better than one
- Author
-
Beverly Wendland and Ruben C. Aguilar
- Subjects
Multidisciplinary ,Epsin ,biology ,Ubiquitin ,media_common.quotation_subject ,Endocytic cycle ,Receptors, Cell Surface ,Biological Sciences ,Endocytosis ,Clathrin ,Cell biology ,Endocytic vesicle ,Caveolae ,Caveolin ,biology.protein ,Humans ,Internalization ,media_common - Abstract
Endocytosis pathways are used by eukaryotic cells for internalization of nutrients, signal transduction regulation, and modulation of plasma membrane composition. The cytoplasmic regions of plasma membrane “cargo” proteins harbor signals that are recognized by “cargo adaptor” proteins; this concentrates the cargo proteins at sites of budding for subsequent internalization via an endocytic vesicle. There are several endocytic pathways that can mediate the internalization of many different cargo proteins. The best-studied pathway is clathrin-dependent endocytosis (CDE), defined by a requirement for the protein clathrin, which is the major component of the endocytic vesicle coat. There are multiple clathrin-independent endocytosis (CIE) pathways that generally depend on cholesterol-rich membrane domains (i.e., rafts, which can also be encased by the protein caveolin to form membrane invaginations called caveolae). Cargo internalization signals are short peptide motifs; yeast also uses posttranslationally conjugated ubiquitin (Ub) moieties (1). In this issue of PNAS, two studies show that Ub can act as an endocytic signal in mammalian cells; in the absence of clathrin, Ub can direct entry via a CIE pathway (2, 3). The idea that ubiquitylated cargo can travel via a CIE pathway was previously unrecognized, probably because the endocytic factors that can recognize and bind Ub (epsin and Eps15) were first shown to play key roles in the CDE pathway. Now, it seems that these endocytic factors may play multiple roles, depending on which signal the cargo protein presents, or which endocytic route is being used. In recent years it …
- Published
- 2005
39. Allosteric effects of monoclonal antibodies on human growth hormone
- Author
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Marie-Catherine Postel-Vinay, Ruben C. Aguilar, Lilia A. Retegui, and Leonor P. Roguin
- Subjects
Models, Molecular ,endocrine system ,medicine.drug_class ,Protein Conformation ,Clinical Biochemistry ,Allosteric regulation ,Monoclonal antibody ,Epitopes ,Antigen ,Allosteric Regulation ,Species Specificity ,Pregnancy ,medicine ,Animals ,Humans ,Binding site ,Rats, Wistar ,Receptor ,Molecular Biology ,Sequence Deletion ,Human liver ,Chemistry ,Human growth hormone ,Antibodies, Monoclonal ,Cell Biology ,General Medicine ,Receptors, Somatotropin ,Molecular biology ,Rats ,Biochemistry ,Growth Hormone ,embryonic structures ,Microsomes, Liver ,Cattle ,Female ,Rabbits ,hormones, hormone substitutes, and hormone antagonists ,Hormone ,Protein Binding - Abstract
We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors. The effect of MAbAE5,AC8 and F11 on hGH binding was measured by determining the formation of 125I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding 125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes. Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case performed 125I-MAb:hGH complexes were added to microsomes. Data showed that 125I-MAb AE5:hGH complexes bound better to the various receptors than 125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to 125I-MAb AC8 or 125I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32-46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding. Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
40. Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes
- Author
-
Andrzej Bartke, Ruben C. Aguilar, Juan M. Dellacha, P. K. Ghosh, R. S. Calandra, Daniel Turyn, and H. N. Fernandez
- Subjects
Genetically modified mouse ,Male ,medicine.medical_specialty ,Somatotropic cell ,Receptors, Prolactin ,Transgene ,Recombinant Fusion Proteins ,Population ,Mice, Transgenic ,Biology ,Mice ,Reference Values ,Internal medicine ,Genes, Regulator ,Genetics ,medicine ,Animals ,Humans ,Bovine somatotropin ,education ,Receptor ,Promoter Regions, Genetic ,education.field_of_study ,Sex Characteristics ,Receptors, Somatotropin ,Prolactin ,Kinetics ,Endocrinology ,Growth Hormone ,Microsomes, Liver ,Animal Science and Zoology ,Cattle ,Female ,Metallothionein ,Phosphoenolpyruvate Carboxykinase (GTP) ,Phosphoenolpyruvate carboxykinase ,Agronomy and Crop Science ,Biotechnology - Abstract
The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.
- Published
- 1992
41. Comparison of Three Techniques for Rapid Determination of Strontium in Soils and Vegetation
- Author
-
Larry E. Wangen, Ruben D. Aguilar, and Ernest S. Gladney
- Subjects
Strontium ,Biochemistry (medical) ,Clinical Biochemistry ,Radiochemistry ,chemistry.chemical_element ,Epithermal neutron ,Biochemistry ,Neutron temperature ,Analytical Chemistry ,law.invention ,chemistry ,law ,Soil water ,Electrochemistry ,medicine ,Irradiation ,medicine.symptom ,Vegetation (pathology) ,Atomic absorption spectroscopy ,Spectroscopy - Abstract
Instrumental epithermal neutron activation analysis, atomic absorption spectrophotometry, and instrumental thermal neutron activation analysis were compared to determine which technique produced best Sr data in the most expeditious fashion. Irradiation with epithermal neutrons was found to be the best method.
- Published
- 1977
42. Interaction of Endocytic Signals from the HIV-1 Envelope Glycoprotein Complex with Members of the Adaptor Medium Chain Family
- Author
-
Hiroshi Ohno, Ruben C. Aguilar, Marie-Christine Fournier, Silke Hennecke, Juan S. Bonifacino, and Pierre Cosson
- Subjects
Recombinant Fusion Proteins ,media_common.quotation_subject ,Endocytic cycle ,Gene Expression ,Nerve Tissue Proteins ,HIV Envelope Protein gp120 ,Biology ,medicine.disease_cause ,Avian Proteins ,Cytosol ,Receptors, HIV ,Glycoprotein complex ,Cell surface receptor ,Virology ,Receptors, Transferrin ,Protein targeting ,medicine ,Humans ,Internalization ,media_common ,Membrane Glycoproteins ,Cell Membrane ,Signal transducing adaptor protein ,Basolateral plasma membrane ,Phosphoproteins ,Clathrin ,HIV Envelope Protein gp41 ,Cell biology ,Adaptor Proteins, Vesicular Transport ,Lymphocyte cytosolic protein 2 ,Monomeric Clathrin Assembly Proteins ,HIV-1 ,HeLa Cells ,Signal Transduction - Abstract
The envelope glycoprotein (Env) complex of HIV-1 undergoes rapid internalization from the plasma membrane of human cells by virtue of a tyrosine-based endocytic signal (RQGYSPL, residues 704-710) in the cytosolic tail of the protein (J. F. Rowell et al., J. Immunol. 155, 473-488, 1995). Here we demonstrate that this tyrosine-based signal interacts with the mu 2 (medium) chain of the AP-2 clathrin-associated adaptor, a protein complex involved in endocytosis of cell surface receptors. The same signal is also capable of interacting with two other members of the adaptor medium chain family, mu 1 and mu 3A, which are components of the AP-1 and AP-3 adaptor complexes, respectively. Interactions with mu 1 and mu 3A might be responsible for the targeting of the internalized envelope glycoprotein to lysosomes or to the basolateral plasma membrane of polarized epithelial cells. A second potential tyrosine-based signal (LFSYHRL, residues 760-766) also interacts with mu 1, mu 2, and mu 3A, although it is less important for internalization in vivo probably due to its position within the cytosolic tail. Overexpression of chimeric proteins having the HIV-1 Env cytosolic tail increases expression of the transferrin receptor on the cell surface, probably due to saturation of the cellular pool of mu 2 by the overexpressed proteins. These observations suggest that HIV-1 Env utilizes the protein sorting machinery of the host cells for internalization and sorting at various steps of the endocytic and biosynthetic pathways.
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43. The medium subunits of adaptor complexes recognize distinct but overlapping sets of tyrosine-based sorting signals
- Author
-
Takashi Saito, David Yeh, Ruben C. Aguilar, Juan S. Bonifacino, Hiroshi Ohno, and Daisuke Taura
- Subjects
Adaptor Protein Complex 3 ,Protein subunit ,Adaptor Protein Complex 1 ,Molecular Sequence Data ,Adaptor Protein Complex 2 ,Nerve Tissue Proteins ,Endosomes ,Protein Sorting Signals ,Biology ,Biochemistry ,Animals ,Amino Acid Sequence ,Tyrosine ,Molecular Biology ,Peptide sequence ,Gene Library ,chemistry.chemical_classification ,Membrane Proteins ,Signal transducing adaptor protein ,Cell Biology ,Phosphoproteins ,Adaptor Protein Complex mu Subunits ,Endocytosis ,Amino acid ,Adaptor Proteins, Vesicular Transport ,chemistry ,Monomeric Clathrin Assembly Proteins ,Vertebrates ,Isoleucine ,Lysosomes - Abstract
Tyrosine-based sorting signals conforming to the motif YXXO (Y is tyrosine, X is any amino acid, and O is an amino acid with a bulky hydrophobic side chain (leucine, isoleucine, phenylalanine, methionine, valine)) interact with the medium (mu) subunits of clathrin adaptor (AP) complexes. We have analyzed the selectivity of interaction between YXXO signals and the mu1, mu2, and mu3 (A or B) subunits of the AP-1, AP-2, and AP-3 complexes, respectively, by screening a combinatorial XXXYXXO library using the yeast two-hybrid system. All the medium subunits were found to prefer proline at position Y+2, suggesting that YXXO signals are stabilized by a bend in the polypeptide backbone. Other than for this common preference, each medium subunit favored specific sets of residues at the X and O positions; these preferences were consistent with the proposed roles of the different adaptor complexes in rapid endocytosis and lysosomal targeting. A considerable specificity overlap was also revealed by these analyses, suggesting that additional factors, such as the context of the signals, must be important determinants of recognition.
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