10 results on '"Noah B. Bloch"'
Search Results
2. The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core
- Author
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Noah B. Bloch, Thomas E. Wales, Michelle S. Prew, Hannah R. Levy, John R. Engen, and Loren D. Walensky
- Subjects
Science - Abstract
The pro-apoptotic BAX protein is a monomer under homeostatic conditions and, in response to stress, transforms into oligomers that induce apoptosis. Here, the authors characterize structural features of BAX that individually stabilize the monomer while collectively contributing to oligomerization.
- Published
- 2021
- Full Text
- View/download PDF
3. Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency
- Author
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Michelle S, Prew, Christina M, Camara, Thomas, Botzanowski, Jamie A, Moroco, Noah B, Bloch, Hannah R, Levy, Hyuk-Soo, Seo, Sirano, Dhe-Paganon, Gregory H, Bird, Henry D, Herce, Micah A, Gygi, Silvia, Escudero, Thomas E, Wales, John R, Engen, and Loren D, Walensky
- Subjects
Mitochondrial Diseases ,Muscular Diseases ,Acyl-CoA Dehydrogenase, Long-Chain ,Congenital Bone Marrow Failure Syndromes ,Humans ,Lipid Metabolism, Inborn Errors - Abstract
Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.
- Published
- 2021
4. The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core
- Author
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Thomas E. Wales, John R. Engen, Noah B. Bloch, Hannah R. Levy, Michelle S. Prew, and Loren D. Walensky
- Subjects
Models, Molecular ,Conformational change ,Protein Conformation ,Science ,Cell ,General Physics and Astronomy ,Apoptosis ,Mitochondrion ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cytosol ,medicine ,Animals ,Humans ,Amino Acids ,Cells, Cultured ,bcl-2-Associated X Protein ,Mice, Knockout ,Binding Sites ,Multidisciplinary ,Mass spectrometry ,Chemistry ,Protein core ,General Chemistry ,Oncogene proteins ,Mitochondria ,Cell biology ,medicine.anatomical_structure ,Mutation ,Helix ,Conformational stability ,Protein Multimerization ,Structural biology ,Protein Binding ,Signal Transduction - Abstract
BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation., The pro-apoptotic BAX protein is a monomer under homeostatic conditions and, in response to stress, transforms into oligomers that induce apoptosis. Here, the authors characterize structural features of BAX that individually stabilize the monomer while collectively contributing to oligomerization.
- Published
- 2021
5. Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks
- Author
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Noah B. Bloch, Craig H. Bassing, Adrian Rivera-Reyes, David G. Schatz, and Megan R. Fisher
- Subjects
0301 basic medicine ,DNA damage ,Immunology ,V(D)J recombination ,chemical and pharmacologic phenomena ,hemic and immune systems ,Biology ,Molecular biology ,Recombination-activating gene ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,chemistry ,Transcription (biology) ,RAG2 ,Immunology and Allergy ,Gene ,Transcription factor ,DNA ,030215 immunology - Abstract
Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double-strand breaks (DSBs) induced at Igκ loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ataxia telangiectasia mutated (ATM) kinase to feedback-inhibit RAG expression and RAG cleavage of the other Igκ allele. In this article, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA, but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic Ag receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of monospecific lymphocytes while suppressing oncogenic Ag receptor locus translocations.
- Published
- 2017
6. Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization
- Author
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Joel C. Bucci, Loren D. Walensky, Lixin Fan, Yael Ben-Nun, Danny Cohen, Steven P. Gygi, Liron David, Catherine E. Newman, Scott B. Hansen, Noah B. Bloch, Edward P. Harvey, Zachary J. Hauseman, John R. Engen, Rida Mourtada, Thomas E. Wales, Julian Mintseris, Devin K. Schweppe, Hao Wu, and Henry D. Herce
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Models, Molecular ,Programmed cell death ,Cell Membrane Permeability ,Protein Conformation ,Apoptosis ,Biology ,Mitochondrion ,Oligomer ,Article ,Protein–protein interaction ,Mice ,chemistry.chemical_compound ,Cytosol ,Animals ,Humans ,Molecular Biology ,bcl-2-Associated X Protein ,Bcl-2 family ,Biological Transport ,Cell Biology ,Mitochondria ,chemistry ,Mitochondrial Membranes ,Biophysics ,Protein Multimerization ,Bacterial outer membrane ,Proto-Oncogene Proteins c-fos ,Alpha helix - Abstract
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAX(O)) for analysis. Here, we report the production and characterization of a full-length BAX(O) that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAX(O) enabled the assignment of specific roles to particular residues and α-helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAX oligomer, revealing structural requirements for the elusive execution-phase of mitochondrial apoptosis.
- Published
- 2020
7. Uncovering protein–protein interactions through a team-based undergraduate biochemistry course
- Author
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Kathleen J. S. Tsai, Louise K. Charkoudian, Emily S. Winesett, Sabina Aliev, Alexandra J. Lamacki, Alexander E. Lafrance, Adam R. Huff, David L. Cookmeyer, Joshua A. Bulos, Christian R. Fagre, Maryna Khromava, Kerilyn N. Godbe, Max J. Cryle, Noah B. Bloch, Robert Fairman, Irene L. Evans, Alana M. Thurston, Jeanne M. Quinn, Bashkim Kokona, Robert C. Parry, Aurelio Mollo, and Daniel M. Konstantinovsky
- Subjects
0301 basic medicine ,Research Facilities ,Interface (Java) ,Social Sciences ,Biochemistry ,Mixed Function Oxygenases ,Learning and Memory ,Spectrum Analysis Techniques ,Cytochrome P-450 Enzyme System ,Materials Physics ,Community Page ,ComputingMilieux_COMPUTERSANDEDUCATION ,Psychology ,Protein Interaction Maps ,Biology (General) ,4. Education ,General Neuroscience ,Physics ,05 social sciences ,050301 education ,Bacterial Biochemistry ,Physical Sciences ,Educational Status ,Curriculum ,General Agricultural and Biological Sciences ,Research Laboratories ,Sedimentation ,Protein Interaction Map ,Human learning ,QH301-705.5 ,Materials Science ,Context (language use) ,Biology ,Research and Analysis Methods ,Original research ,Microbiology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Human Learning ,Learning ,Humans ,Protein Interactions ,Students ,General Immunology and Microbiology ,Research ,Teaching ,Undergraduate education ,Cognitive Psychology ,Biology and Life Sciences ,Proteins ,Bacteriology ,030104 developmental biology ,People and Places ,Cognitive Science ,Ultraviolet-Visible Spectroscopy ,Population Groupings ,Laboratories ,0503 education ,Undergraduates ,Neuroscience - Abstract
How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.
- Published
- 2017
8. Synthesis and Evaluation of Cytosolic Phospholipase A2 Activatable Fluorophores for Cancer Imaging
- Author
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Michael G. Chiorazzo, Anatoliy V. Popov, Edward J. Delikatny, and Noah B Bloch
- Subjects
Fluorophore ,Biomedical Engineering ,Pharmaceutical Science ,Triple Negative Breast Neoplasms ,Bioengineering ,Phospholipase ,Article ,Fluorescence spectroscopy ,chemistry.chemical_compound ,Cytosol ,Phospholipase A2 ,Cell Line, Tumor ,Phosphatidylcholine ,Fluorescence microscope ,Humans ,Enzyme Assays ,Fluorescent Dyes ,Pharmacology ,Phospholipase A ,Arachidonic Acid ,biology ,Optical Imaging ,Organic Chemistry ,Phospholipases A2 ,chemistry ,Biochemistry ,biology.protein ,Acridines ,Female ,Arachidonic acid ,Biotechnology - Abstract
Activatable fluorophores selective to cytosolic phospholipase A2 (cPLA2) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA2 was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA2 when compared to esterification with palmitic acid. In vitro analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a non-selective phospholipase (sPLA2 group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally in vitro and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA2. In contrast to the in vitro analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA2 enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA2 resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA2 selective activatable fluorophore capable of detecting cPLA2 in triple negative breast cancer cells.
- Published
- 2015
9. Immature Lymphocytes Inhibit
- Author
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Megan R, Fisher, Adrian, Rivera-Reyes, Noah B, Bloch, David G, Schatz, and Craig H, Bassing
- Subjects
Homeodomain Proteins ,Mice, Knockout ,Transcription, Genetic ,Blotting, Western ,chemical and pharmacologic phenomena ,hemic and immune systems ,Lymphoid Progenitor Cells ,Flow Cytometry ,Real-Time Polymerase Chain Reaction ,V(D)J Recombination ,Article ,DNA-Binding Proteins ,Mice, Inbred C57BL ,Blotting, Southern ,Mice ,Gene Expression Regulation ,Animals ,DNA Breaks, Double-Stranded - Abstract
Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double strand breaks (DSBs) induced at Igκ loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ATM kinase to feedback inhibit RAG expression and RAG cleavage of the other Igκ allele. Here, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic antigen receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of mono-specific lymphocytes while suppressing oncogenic antigen receptor locus translocations.
- Published
- 2016
10. Uncovering protein-protein interactions through a team-based undergraduate biochemistry course.
- Author
-
David L Cookmeyer, Emily S Winesett, Bashkim Kokona, Adam R Huff, Sabina Aliev, Noah B Bloch, Joshua A Bulos, Irene L Evans, Christian R Fagre, Kerilyn N Godbe, Maryna Khromava, Daniel M Konstantinovsky, Alexander E Lafrance, Alexandra J Lamacki, Robert C Parry, Jeanne M Quinn, Alana M Thurston, Kathleen J S Tsai, Aurelio Mollo, Max J Cryle, Robert Fairman, and Louise K Charkoudian
- Subjects
Biology (General) ,QH301-705.5 - Abstract
How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.
- Published
- 2017
- Full Text
- View/download PDF
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