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The extracellular signal-regulated kinase (ERK1/2) pathway is essential in embryonic development. The scaffold protein Shoc2 is a critical modulator of ERK1/2 signals, and mutations in the shoc2 gene lead to the human developmental disease known as Noonan-like syndrome with loose anagen hair (NSLH). The loss of Shoc2 and the shoc2 NSLH-causing mutations affect the tissues of neural crest (NC) origin. In this study, we utilized the zebrafish model to dissect the role of Shoc2-ERK1/2 signals in the development of NC. These studies established that the loss of Shoc2 significantly altered the expression of transcription factors regulating the specification and differentiation of NC cells. Using comparative transcriptome analysis of NC-derived cells from shoc2 CRISPR/Cas9 mutant larvae, we found that Shoc2-mediated signals regulate gene programs at several levels, including expression of genes coding for the proteins of extracellular matrix (ECM) and ECM regulators. Together, our results demonstrate that Shoc2 is an essential regulator of NC development. This study also indicates that disbalance in the turnover of the ECM may lead to the abnormalities found in NSLH patients.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

The ERK1/2 (also known as MAPK3 and MAPK1, respectively) signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins, such as Shoc2 to guide and fine-tune its signals. Mutations in SHOC2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here, we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We reveal that, in the Shoc2 module, USP7 functions as a molecular ‘switch’ that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH, thereby extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.

Valosin-containing protein (VCP), also named p97, is an essential hexameric AAA+ ATPase with diverse functions in the ubiquitin system. Here we demonstrate that VCP is critical in controlling signals transmitted via the essential Shoc2-ERK1/2 signaling axis. The ATPase activity of VCP modulates the stoichiometry of HUWE1 in the Shoc2 complex as well as HUWE1-mediated allosteric ubiquitination of the Shoc2 scaffold and the RAF-1 kinase. Abrogated ATPase activity leads to augmented ubiquitination of Shoc2/RAF-1 and altered phosphorylation of RAF-1. We found that in fibroblasts from patients with inclusion body myopathy with Paget’s disease of bone and frontotemporal dementia (IBMPFD) that harbor germline mutations in VCP, the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced. This study provides a mechanistic basis for the critical role of VCP in the regulation of the ERK1/2 pathway and reveals a previously unrecognized function of the ERK1/2 pathway in the pathogenesis of IBMPFD.

The extracellular signal-related kinase 1 and 2 (ERK1/2) pathway is a highly conserved signaling cascade with numerous essential functions in development. The scaffold protein Shoc2 amplifies the activity of the ERK1/2 pathway and is an essential modulator of a variety of signaling inputs. Germline mutations in Shoc2 are associated with the human developmental disease known as the Noonan-like syndrome with loose anagen hair. Clinical manifestations of this disease include congenital heart defects, developmental delays, distinctive facial abnormalities, reduced growth and cognitive deficits along with hair anomalies. The many molecular details of pathogenesis of the Noonan-like syndrome and related developmental disorders, cumulatively called RASopathies, remain poorly understood. Mouse knockouts for Shoc2 are embryonic lethal, emphasizing the need for additional animal models to study the role of Shoc2 in embryonic development. Here, we characterize a zebrafish shoc2 mutant, and show that Shoc2 is essential for development, and that its loss is detrimental for the development of the neural crest and for hematopoiesis. The zebrafish model of the Noonan-like syndrome described here provides a novel system for the study of structure–function analyses and for genetic screens in a tractable vertebrate system.

Pancreatology Club Professional Medical Community, 1A.S. Loginov Moscow Clinical Research and Practical Center, Moscow Healthcare Department, Moscow; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Ministry of Health of Russia, Moscow; 3Kazan State Medical University, Ministry of Health of Russia, Kazan; 4Kazan (Volga) Federal University, Kazan; 5Far Eastern State Medical University, Ministry of Health of Russia, Khabarovsk; 6Morozov City Children's Clinical Hospital, Moscow Healthcare Department, Moscow; 7I.I. Mechnikov North-Western State Medical University, Ministry of Health of Russia, Saint Petersburg; 8Siberian State Medical University, Ministry of Health of Russia, Tomsk; 9M.F. Vladimirsky Moscow Regional Research Clinical Institute, Moscow; 10Maimonides State Classical Academy, Moscow; 11V.I. Razumovsky State Medical University, Ministry of Health of Russia, Saratov; 12I.M. Sechenov First Moscow State Medical University, Ministry of Health of Russia, Moscow; 13S.M. Kirov Military Medical Academy, Ministry of Defense of Russia, Saint Petersburg; 14Surgut State Medical University, Ministry of Health of Russia, Surgut; 15City Clinical Hospital Five, Moscow Healthcare Department, Moscow; 16Nizhny Novgorod Medical Academy, Ministry of Health of Russia, Nizhny Novgorod; 17Territorial Clinical Hospital Two, Ministry of Health of the Krasnodar Territory, Krasnodar; 18Saint Petersburg State Pediatric Medical University, Ministry of Health of Russia, Saint Petersburg; 19Rostov State Medical University, Ministry of Health of Russia, Rostov-on-Don; 20Omsk Medical University, Ministry of Health of Russia, Omsk; 21Russian Medical Academy of Postgraduate Education, Ministry of Health of Russia, Moscow; 22Novosibirsk State Medical University, Ministry of Health of Russia, Novosibirsk; 23Stavropol State Medical University, Ministry of Health of Russia, Stavropol; 24Kemerovo State Medical University, Ministry of Health of Russia, Kemerovo; 25N.I. Pirogov Russian National Research Medical University, Ministry of Health of Russia, Moscow; 26A.M. Nikiforov All-Russian Center of Emergency and Radiation Medicine, Russian Ministry for Civil Defense, Emergencies and Elimination of Consequences of Natural Disasters, Saint Petersburg; 27Research Institute for Medical Problems of the North, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk; 28S.P. Botkin City Clinical Hospital, Moscow Healthcare Department, Moscow; 29Tver State Medical University, Ministry of Health of Russia, Tver The Russian consensus on the diagnosis and treatment of chronic pancreatitis has been prepared on the initiative of the Russian Pancreatology Club to clarify and consolidate the opinions of Russian specialists (gastroenterologists, surgeons, and pediatricians) on the most significant problems of diagnosis and treatment of chronic pancreatitis. This article continues a series of publications explaining the most significant interdisciplinary consensus statements and deals with enzyme replacement therapy.Российский консенсус по диагностике и лечению хронического панкреатита подготовлен по инициативе Российского 'Панкреатологического клуба' с целью выяснения и консолидации мнений отечественных специалистов (гастроэнтерологов, хирургов и педиатров) по наиболее важным проблемам диагностики и лечения хронического панкреатита. Настоящая статья продолжает серию публикаций, разъясняющих наиболее важные положения междисциплинарного консенсуса и посвящена вопросам заместительной ферментной терапии.

The accurate transmission of signals by the canonical ERK1/2 kinase pathway critically relies on the proper assembly of an intricate multiprotein complex by the scaffold protein Shoc2. However, the details of the mechanism by which Shoc2 guides ERK1/2 signals are not clear, in part, due to the lack of research tools targeting specific protein binding moieties of Shoc2. We report generation and characterization of single domain antibodies against human Shoc2 using a universal synthetic library of humanized nanobodies. Our results identify eight synthetic single-domain antibodies and show that two evaluated antibodies have binding affinities to Shoc2 in the nanomolar range. High affinity antibodies were uniquely suited for the analysis of the Shoc2 complex assembly. Selected single-domain antibodies were also functional in intracellular assays. This study illustrates that Shoc2 single-domain antibodies can be used to understand functional mechanisms governing complex multiprotein signaling modules and have promise in application for therapies that require modulation of the ERK1/2-associated diseases.

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax. The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.

Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide - 11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and d-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function.

The extracellular signal-regulated kinase (ERK1/2) cascade regulates a myriad of functions in multicellular organisms. Scaffold proteins provide critical spatial and temporal control over the specificity of signaling. Shoc2 is a scaffold that accelerates activity of the ERK1/2 pathway. Loss of Shoc2 expression in mice results in embryonic lethality, thus highlighting the essential role of Shoc2 in embryogenesis. In agreement, patients carrying mutated Shoc2 suffer from a wide spectrum of developmental deficiencies. Efforts to understand the mechanisms by which Shoc2 controls ERK1/2 activity revealed the intricate machinery that governs the ability of Shoc2 to transduce signals of the ERK1/2 pathway. Understanding the mechanisms by which Shoc2 contributes to a high degree of specificity of ERK1/2 signaling as well as deciphering the biological functions of Shoc2 in development and human disorders are major unresolved questions.

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway plays a central role in defining various cellular fates. Scaffold proteins modulating ERK1/2 activity control growth factor signals transduced by the pathway. Here, we analyzed signals transduced by Shoc2, a critical positive modulator of ERK1/2 activity. We found that loss of Shoc2 results in impaired cell motility and delays cell attachment. As ERKs control cellular fates by stimulating transcriptional response, we hypothesized that the mechanisms underlying changes in cell adhesion could be revealed by assessing the changes in transcription of Shoc2-depleted cells. Using quantitative RNA-seq analysis, we identified 853 differentially expressed transcripts. Characterization of the differentially expressed genes showed that Shoc2 regulates the pathway at several levels, including expression of genes controlling cell motility, adhesion, crosstalk with the transforming growth factor beta (TGFβ) pathway, and expression of transcription factors. To understand the mechanisms underlying delayed attachment of cells depleted of Shoc2, changes in expression of the protein of extracellular matrix (lectin galactoside-binding soluble 3-binding protein; LGALS3BP) were functionally analyzed. We demonstrated that delayed adhesion of the Shoc2-depleted cells is a result of attenuated expression and secretion of LGALS3BP. Together our results suggest that Shoc2 regulates cell motility by modulating ERK1/2 signals to cell adhesion.

To study spatiotemporal regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling cascade in living cells, a HeLa cell line in which MAPK kinase of ERK kinase (MEK) 2 (MAPK kinase) was knocked down by RNA interference and replaced with the green fluorescent protein (GFP)-tagged MEK2 was generated. In these cells, MEK2–GFP was stably expressed at a level similar to that of the endogenous MEK2 in the parental cells. Upon activation of the EGF receptor (EGFR), a pool of MEK2–GFP was found initially translocated to the plasma membrane and then accumulated in a subset of early and late endosomes. However, activated MEK was detected only at the plasma membrane and not in endosomes. Surprisingly, MEK2–GFP endosomes did not contain active EGFR, suggesting that endosomal MEK2–GFP was separated from the upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in increased activation of ERK without affecting the activity of MEK2–GFP. The accumulation of MEK2–GFP in endosomes was also blocked by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the negative feedback regulation of the EGFR–MAPK signaling pathway by endocytosis.

Restoration of bile flow after 9-day cholestasis in rat liver normalized the content of lipid peroxidation products. The removal of the cholestatic factor after 12-day cholestasis was not followed by recovery of these parameters. We showed that measurement of serum concentration of lipid peroxidation products in patients with cholelithiasis during the preoperative period holds promise for selection of the optimum time for surgical treatment and prediction of the risk of postoperative complications.

The scaffold protein Shoc2 accelerates activity of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) pathway. Mutations in Shoc2 result in Noonan-like RASopathy, a developmental disorder with a wide spectrum of symptoms. The amplitude of the ERK1/2 signals transduced through the complex is fine-tuned by the HUWE1-mediated ubiquitylation of Shoc2 and its signaling partner RAF-1. Here, we provide a mechanistic basis of how ubiquitylation of Shoc2 and RAF-1 is controlled. We demonstrate that the newly identified binding partner of Shoc2, the (AAA+) ATPase PSMC5, triggers translocation of Shoc2 to endosomes. At the endosomes, PSMC5 displaces the E3 ligase HUWE1 from the scaffolding complex to attenuate ubiquitylation of Shoc2 and RAF-1. We show that a RASopathy mutation that changes the subcellular distribution of Shoc2 leads to alterations in Shoc2 ubiquitylation due to the loss of accessibility to PSMC5. In summary, our results demonstrate that PSMC5 is a new and important player involved in regulating ERK1/2 signal transmission through the remodeling of Shoc2 scaffold complex in a spatially-defined manner.

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.

Changes in sphingomyelinase activity, tumor necrosis factor α expression, and level of lipid peroxidation products in the course of development of cholestatic liver injury have been studied. The same type phase shifts in the analyzed parameters were observed, which included a marked decrease at the early stages of cholestasis (days 3–6) and a pronounced increase at the later stages (days 12–16), i.e., under the conditions of developed pathology. There was a significant positive linear correlation between tumor necrosis factor α expression, sphingomyelinase activity, and lipid peroxidation intensity in cholestatic injury. We propose that detected changes may reflect the balance between the effects of the two major bile components—bilirubin, which is accumulated in the liver at the early stages of cholestasis, and bile acids, whose influence dominates at the later stages of pathologic process. Our results indicate that tumor necrosis factor α overexpression, sphingomyelin cycle activation, and lipid peroxidation intensification may cause apoptosis of hepatocytes at the late stages of cholestasis.

Here we report the characterization of an eps15 homology (EH) domain containing protein designated EHD3. EHD3 was mapped to human chromosome 2p22–23, while the murine Ehd3 homolog was mapped to chromosome 17p21. Both the human and the mouse genes contain a polymorphic (CA) repeat in their 3′UTR. One 3.6-kb Ehd3 transcript was mainly detected in adult mouse brain and kidney and at day 7 of mouse development. On the other hand, human tissues exhibited two, 4.2- and 3.6-kb, EHD3 RNA species. They were predominantly expressed in heart, brain, placenta, liver, kidney and ovary. EHD3, expressed as a green fluorescent fusion protein was localized to endocytic vesicles and to microtubule-dependent, membrane tubules. There was a clear colocalization of EHD3-positive structures and transferrin-containing recycling vesicles, implying that EHD3 resides within the endocytic recycling compartment. Shuffling the N-terminal domain of EHD1 (previously shown to reside in the transferrin-containing, endocytic recycling compartment) with that of EHD3 resulted in a chimeric EHD protein that was localized mainly to tubules instead of the endocytic vesicles, implicating the N-terminal domain as responsible for the tubular localization of EHD3. Mutant EHD3 forms, missing the N-terminal or the C-terminal domains, lost their tubular localization. Results of two-hybrid analyses indicated that EHD1 and EHD3 interact with each other. In addition, EHD1 and EHD3 could be coimmunoprecipitated from cellular extracts, confirming the interaction implied by two-hybrid analysis. Moreover, coexpression of EHD1 and EHD3 resulted in their colocalization in microtubule-dependent tubules as well as in punctate forms. Based on its specific intracellular localization and its interaction with EHD1, we postulate that EHD3 localizes on endocytic tubular and vesicular structures and regulates their microtubule-dependent movement.

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF-alpha secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-alpha, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF-alpha detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF-alpha is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.

Rasopathies are a group of genetic disorders caused by germline mutations in multiple genes of the Extracellular signal-Regulated Kinases 1 and 2 (ERK1/2) pathway. The only previously identified missense mutation in SHOC2, a scaffold protein of the ERK1/2 pathway, led to Noonan-like syndrome with loose anagen hair. Here we report a novel mutation in SHOC2(c.519G>A; p.M173I) that leads to a Rasopathy with clinical features partially overlapping those occurring in Noonan and Cardio-Facio-Cutaneous syndromes. Studies to clarify the significance of this SHOC2 variant revealed that the mutant protein has impaired capacity to interact with protein phosphatase 1c (PP1c), leading to insufficient activation of RAF-1 kinase. This SHOC2 variant thus is unable to fully rescue ERK1/2 activity in cells depleted of endogenous SHOC2. We conclude that SHOC2 mutations can cause a spectrum of Rasopathy phenotypes in heterozygous individuals. Importantly, our work suggests that individuals with mild Rasopathy symptoms may be under-diagnosed.

Spatial distribution of intracellular signaling molecules and assembly of signaling complexes are yet to be fully understood. Studies of signaling events in time or space present a particular challenge due to the adverse effects that overexpression of signaling proteins may have on their functions and localization. To follow the distribution of signaling proteins in living cells we developed a methodology named knockdown and reconstitution (KDAR) that allows one to visualize proteins at levels of expression that are close to physiological. This methodology provides a stable expression of “endogenous” shRNA for long-term silencing of the targeted gene and simultaneous expression of a DNA cassette coding for a fluorescently labeled protein, which is insensitive to the targeting shRNA. In this chapter we discuss the needed reagents and outline two experimental approaches to generate KDAR stable cell lines. First, we demonstrate how the plasmid-mediated KDAR approach is successfully utilized to visualize spatial distribution of the GFP-labeled MEK2 in living cells. We then show how the lentivirus-mediated KDAR approach is used to reconstitute and visualize expression of the ERK1/2 scaffold protein Shoc2.

Background/Aim: Pyridoxine deficiency may be the cause of failure to respond appropriately to iron and erythropoietin (EPO) administration in hemodialysis patients. Method: We studied 36 patients on chronic hemodialysis amply supplemented with iron and EPO, who failed to raise hematocrit levels >33%. Patients were divided into three equal groups and evaluated for 6 months as follows: Group A – no additional therapy; group B – supplemented with oral pyridoxine 50 mg/day, and group C received 100 mg/day pyridoxine orally. Results: In all our patients, erythrocyte pyridoxine levels were initially within reference range for a healthy population and did not vary significantly during the study period. Likewise, ferritin levels and iron saturation values remained normal and constant. Hemoglobin and/or hematocrit levels remained practically unchanged in all three groups. Conclusions: The results indicate that in hemodialysis patients with normal pyridoxine status who, despite appropriate supplementation of iron and EPO, fail to reach optimal hematocrit levels, additional pyridoxine treatment does not produce any hematocrit elevation.

Bardet–Biedl Syndrome (BBS) is an autosomal recessive disorder characterized by developmental abnormalities including mental retardation, obesity, retinitis pigmentosa, polydactyly, short stature, and hypogenitalism. To date, five BBS loci have been identified. BBS1, located on 11q13, is reported to be the most prevalent form of BBS in the Caucasian population. A positional cloning approach is being used to identify the gene responsible for BBS1. EHD1, a new member of the EH-domain containing proteins, was identified in this study as lying within the BBS1 disease interval. RNA analysis of many tissues revealed that expression of EHD1 is ubiquitous, with elevated levels in the testis. The genomic structure of EHD1 was elucidated by direct BAC sequencing. Following identification of the intron/exon boundaries, mutational analysis was performed by single strand conformation polymorphism and direct sequencing of affected individuals from several large kindreds linked to the BBS1 locus, as well as a cohort of unrelated probands. No disease-causing mutations were identified in this analysis, but several polymorphisms were found.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.

Gaucher disease, the most prevalent sphingolipid disorder, is characterized by an accumulation of sphingolipids mainly in cells of the reticuloendothelial cells, and is due to decreased activity of the lysosomal enzyme glucocerebrosidase (GCase). The corresponding gene is expressed differentially, namely in different cell types there are different GCase steady-state mRNA levels. Electrophoretic mobility shift assays, DNase footprinting and chloramphenicol acetyl transferase assays were employed in order to unravel some of the transcription factors responsible for the differential expression of the glucocerebrosidase (gcs) gene. The results indicate that OCTA binding protein, AP-1, PEA3 and a CAAT binding protein participate in regulating the expression of the gcs gene. The availability of the transcription factors seems to control the level of transcription of the gcs gene.

Spatial distribution of intracellular signaling molecules and assembly of signaling complexes are yet to be fully understood. Studies of signaling events in time or space present a particular challenge due to the adverse effects that overexpression of signaling proteins may have on their functions and localization. To follow the distribution of signaling proteins in living cells we developed a methodology named knockdown and reconstitution (KDAR) that allows one to visualize proteins at levels of expression that are close to physiological. This methodology provides a stable expression of "endogenous" shRNA for long-term silencing of the targeted gene and simultaneous expression of a DNA cassette coding for a fluorescently labeled protein, which is insensitive to the targeting shRNA. In this chapter we discuss the needed reagents and outline two experimental approaches to generate KDAR stable cell lines. First, we demonstrate how the plasmid-mediated KDAR approach is successfully utilized to visualize spatial distribution of the GFP-labeled MEK2 in living cells. We then show how the lentivirus-mediated KDAR approach is used to reconstitute and visualize expression of the ERK1/2 scaffold protein Shoc2.

A new surgical method of treating patients with unstable insulin-dependent diabetes (IDD) has been developed-that of surgically shunting pancreatic blood into the systemic blood flow with the purpose of creating a more optimal interaction of subcutaneously administered insulin and pancreas-secreted glucagon.The long term results of the operation depend on the patency of a splenorenal anastomosis. This has been studied by following up 137 patients over periods from half a year to three years. Anastomotic patency was determined by renal and splenic venography and celiacy arteriography, which revealed a patent anastomosis in 114 patients, and an obliterated one in 23.Patients with patent anastomoses showed a lowering of glycosylated hemoglobin (HbAlc) from 13.3±0.03% to 9.3±0.6%, p2, from 49.2±2.4 to 58.1±1.9 mm Hg, plc levels did not change from preoperative values: 12.9±0.4% and 12.8±0.7%, pThe suggested method is not an alternative for insulin therapy, but considerably enhances its potential.

Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.

Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP‐bound conformation in living cells. Rab5‐binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5‐tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP‐Rab5 and YFP‐Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP‐bound Rab5. GTP‐bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three‐chromophore FRET (3‐FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3‐FRET analysis suggest that GTP‐bound Rab5 is capable of oligomerization and present in multiprotein complexes.

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.

Here we report the characterization of an eps15 homology (EH) domain containing protein designated EHD3. EHD3 was mapped to human chromosome 2p22-23, while the murine Ehd3 homolog was mapped to chromosome 17p21. Both the human and the mouse genes contain a polymorphic (CA) repeat in their 3'UTR. One 3.6-kb Ehd3 transcript was mainly detected in adult mouse brain and kidney and at day 7 of mouse development. On the other hand, human tissues exhibited two, 4.2- and 3.6-kb, EHD3 RNA species. They were predominantly expressed in heart, brain, placenta, liver, kidney and ovary. EHD3, expressed as a green fluorescent fusion protein was localized to endocytic vesicles and to microtubule-dependent, membrane tubules. There was a clear colocalization of EHD3-positive structures and transferrin-containing recycling vesicles, implying that EHD3 resides within the endocytic recycling compartment. Shuffling the N-terminal domain of EHD1 (previously shown to reside in the transferrin-containing, endocytic recycling compartment) with that of EHD3 resulted in a chimeric EHD protein that was localized mainly to tubules instead of the endocytic vesicles, implicating the N-terminal domain as responsible for the tubular localization of EHD3. Mutant EHD3 forms, missing the N-terminal or the C-terminal domains, lost their tubular localization. Results of two-hybrid analyses indicated that EHD1 and EHD3 interact with each other. In addition, EHD1 and EHD3 could be coimmunoprecipitated from cellular extracts, confirming the interaction implied by two-hybrid analysis. Moreover, coexpression of EHD1 and EHD3 resulted in their colocalization in microtubule-dependent tubules as well as in punctate forms. Based on its specific intracellular localization and its interaction with EHD1, we postulate that EHD3 localizes on endocytic tubular and vesicular structures and regulates their microtubule-dependent movement.

Twenty-two of 24 hemodialysis patients dialyzed simultaneously with a new batch of cellulose acetate dialyzers promptly developed a spectrum of symptoms and physical signs including red eyes, hearing loss, tinnitus, and bone pain, previously described as red eye syndrome. We subsequently injected 4 rabbits with an eluate from a dialyzer of the same or a control batch. Six hours following exposure, the animals developed, in addition to red eyes, diffuse eosinophilic infiltration of various organs as well as myopathic changes and moderate brain edema. On the basis of these data, we suggest that it cannot be concluded whether the underlying pathophysiological mechanisms were toxic, allergic or both and that the occurrence of relevant symptomatology in 2 or more simultaneously dialyzed patients is a strong argument against unnecessary diagnostic or therapeutic procedures. Finally, a second exposure in a given patient should be avoided.

Ligand-induced receptor-mediated endocytosis plays a central role in regulating signaling conveyed by tyrosine kinase receptors. This process depends on the recruitment of the adaptor protein 2 (AP-2) complex, clathrin, dynamin, and other accessory proteins to the ligand-bound receptor. We show here that besides AP-2 and clathrin, two other proteins participate in the endocytic process of the insulin-like growth factor receptor (IGF-1R); they are EHD1, an Eps15 homology (EH) domain-containing protein 1, and SNAP29, a synaptosomal-associated protein. EHD1 and SNAP29 form complexes with alpha-adaptin of AP-2 and co-localize in endocytic vesicles, indicating a role for them in endocytosis. EHD1 and SNAP29 interact directly with each other and are present in complexes with IGF-1R. After IGF-1 induction, EHD1 and IGF-1R co-localize intracellularly. Overexpression of EHD1 in Chinese hamster ovary cells represses IGF-1-mediated signaling, as measured by mitogen-activated protein kinase phosphorylation and Akt phosphorylation, indicating that EHD1 plays a role as a down-regulator in IGF-1 signaling pathway.

While the bulk of renal hypertrophy induced by contralateral nephrectomy or a high-protein diet consists of tubular cell growth, there is some evidence suggesting that mesangial cells play a role in this phenomenon. Previous data suggest that this role of mesangial cells is associated with their proliferation. We, therefore, undertook this investigation to assess the proliferative responses of mesangial cells, originating from single remaining kidneys or from kidneys of rats fed a high-protein diet, to epinephrine, endothelin, arginine vasopressin, neo-synephrine, or epidermal growth factor (EGF). All agents significantly enhanced the proliferation of normal mesangial cells, though the responses to neo-synephrine and EGF were significantly lower as compared with the other growth promoters. The mitogenic effects of the first three agents on single kidney mesangial cells were significant, but blunted as compared with control cells. This blunting was not evident in the case of the latter two mitogens. A significant enhancement of proliferation of mesangial cells originating from protein-fed rats was produced by epinephrine, neo-synephrine, and EGF. These effects were statistically not different from those observed in normal mesangial cells. The proliferative response to each of the mitogens used in the study proved highly specific for each mitogen, since it was abolished by respective specific inhibitors. Mesangial cells may play a role in the activation and later in progressive inhibition of renal hypertrophy in vivo.

The Suppressor of Clear, Caenorhabditis elegans Homolog (SHOC2) is a scaffold protein that positively modulates activity of the RAS/ERK1/2 MAP kinase signaling cascade. We set out to understand the ERK1/2 pathway transcriptional response transduced through the SHOC2 scaffolding module. This data article describes raw gene expression within triplicates of kidney fibroblast-like Cos1 cell line expressing non-targeting shRNA (Cos-NT) and triplicates of Cos1 cells depleted of SHOC2 using shRNA (Cos-LV1) upon activation of ERK1/2 pathway by the Epidermal Growth Factor Receptor (EGFR). The data referred here is available in NCBI׳s Gene Expression Omnibus (GEO), accession GEO: GSE67063 as well as NCBI׳s Sequence Read Archive (SRA), accession SRA: SRP056324. A complete analysis of the results can be found in “Shoc2-tranduced ERK1/2 motility signals – Novel insights from functional genomics”(Jeoung et al., 2016) [1].