SH3 Domain Research Articles

Structural insights and computational molecular docking to explore novel therapeutic drug targets of STAT3

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor, that contains a DNA-binding domain, N-terminal domain, and SH2 domain. The dysregulation of STAT3 activity has been associated with various diseases, such as chronic inflammation and autoimmune disorders. In cancer, STAT3 is often constitutively activated and promotes tumor cell survival, proliferation, and immune evasion. Various bioinformatics approaches were employed to predict the 3D structure of STAT3, followed by a comprehensive evaluation of the predicted model. 3D predicted structure of the target protein revealed an overall quality factor of 94. 45%. It was also observed through the Ramachandran plot that 1.26% residues of the predicted structure of STAT3 were present in the outlier region of the protein structure. Computational docking studies were done to identify the novel drug targets against STAT3. The screened compound via high throughput virtual screening may have the potential to regulate the activity of STAT3. The lowest binding energy of -8.7 Kcal/mol was observed. His-457, Tyr-456, Lys-488, Pro-487, Gln-326, Leu-459, Lys-244, Gln-247 conserved residues were observed. The structural insight and functional determination of STAT3 depend on the identification of the potent binding domain in protein 3D structure.

Screening the Complex Biological Behavior of Late Lanthanides through Genome-wide Interactions.

Despite their similar physicochemical properties, recent studies have demonstrated that lanthanides can display different biological behaviors. Hence, the lanthanide series can be divided into three parts, namely early, mid and late lanthanides, based on their interactions with biological systems. In particular, the late lanthanides demonstrate distinct, but poorly-understood biological activity. In the current study, we employed genome-wide functional screening to help understand biological effects of exposure to Yb(III) and Lu(III), which were selected as representatives of the late lanthanides. As a model organism, we used Saccharomyces cerevisiae, since it shares many biological functions with humans. Analysis of the functional screening results indicated toxicity of late lanthanides is consistent with disruption of vesicle-mediated transport, and further supported a role for calcium transport processes and mitophagy in mitigating toxicity. Unexpectedly, our analysis suggested that late lanthanides target proteins with SH3 domains, which may underlie the observed toxicity. This study provides fundamental insights into the unique biological chemistry of late lanthanides, which may help devise new avenues towards the development of decorporation strategies and bio-inspired separation processes.

Regulation of microRNA expression by the adaptor protein GRB2

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.

Hematopoietic progenitor kinase 1 inhibits the development and progression of pancreatic intraepithelial neoplasia.

Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.

NMR approaches to study proteins integrating globular and disordered domains: the case of c-Src

Abstract Nuclear Magnetic Resonance is one of the most versatile structural biology tools. Its unique capacities remain unchallenged by the advances in other techniques, experimental, like cryo-electron microscopy, or computational, such as AlphaFold. In this perspective article we present the role played by various NMR techniques in the study of c-Src, a non-receptor protein tyrosine kinase that contains globular and intrinsically disordered domains. We show (i) how NMR helped chemical biology to discover the regulatory role of the Unique domain, (ii) its role in the characterization of the fuzzy intramolecular complex connecting the disordered region with the globular core through the SH3 domain, (iii) the identification of salt bridges connecting the main post-translational sites of the Unique domain with neighbor basic residues, and, (iv) the characterization of breathing motions and the independent dynamics of the two lobes of the kinase domain.

Improved methodology for protein NMR structure calculation using hydrogen bond restraints and ANSURR validation: The SH2 domain of SH2B1

Protein structures calculated using NMR data are less accurate and less well-defined than they could be. Here we use the program ANSURR to show that this deficiency is at least in part due to a lack of hydrogen bond restraints. We describe a protocol to introduce hydrogen bond restraints into the structure calculation of the SH2 domain from SH2B1 in a systematic and transparent way and show that the structures generated are more accurate and better defined as a result. We also show that ANSURR can be used as a guide to know when the structure calculation is good enough to stop.

Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

A Model of Interaction Between Apocynin and NADPH Oxidase Enzyme to Analyze the Possible Targets Responsible for Inhibition by Computational Analysis

Background/Aims: A multi-component enzyme system called NADPH oxidase (NOX) helps innate immunity by generating reactive oxygen species (ROS). NOX hyperactivation has been associated w several diseases. This enzyme is a membrane-bound complex made up of six subunits when it is active. These enzymatic subunits are considered to be potent inhibitors of enzyme activity and good targets for reducing oxidative stress. Methods: The present study aimed to analyze the possible targets: the different subunits of NOX, for their interactions with apocynin to identify its possible mechanism of inhibition for NOX, using in silico tools. Monomer, dimer, and trimer of apocynin were docked to various subunits of NOX. Results: Comparable glide scores were obtained when the monomer and dimer of apocynin were docked with p47phox complete subunit of NOX and were better than in comparison to trimer. Free Energy of Binding (FEB) was highest in the case of the trimer (-37.4 Kcal/mol), followed by the dimer (-21.2 Kcal/mol) and monomer (-18.2 Kcal/mol). Dimer obtained the highest glide score of 8.25 (FEB =-25.1 Kcal/mol) with p67phox-isoform 2. The PH domain of p47phox and the SH3 domain of p67phox have their own best binding energy with dimmer. While molecular docking with Rac-Zn-GD, P, dimer, and trimer have shown comparable FEB. The residues, on which the ligands were found to interact, were of major significance being present in those domains that vicinity to inhibit or activate the complex and are important for the protein structure and functioning. MDS studies have confirmed the findings that the Apocynin trimer molecule has superior stability and interactions with the enzyme complex. Conclusion: It can be concluded from the study that trimer and dimer have better interactions in terms of FEB with p67phox and p47phox, indicating the reported findings in the literature.

Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions

RNA binding motif protein X‐linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene’s importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Magnolol as STAT3 inhibitor for treating multiple sclerosis by restricting Th17 cells

ObjectiveMultiple sclerosis (MS) is an immune disease in the central nervous system (CNS) associated with Th17 cells. Moreover, STAT3 initiates Th17 cell differentiation and IL-17A expression through facilitating RORγt in MS. Here, we reported that magnolol, isolated from Magnolia officinalis Rehd. Et Wils, was regarded as a candidate for MS treatment verified by both in vitro and in vivo studies. MethodsIn vivo, experimental autoimmune encephalomyelitis (EAE) model in mice was employed to evaluate the alleviation of magnolol on myeloencephalitis. In vitro, FACS assay was employed to evaluate the effect of magnolol on Th17 and Treg cell differentiation and IL-17A expression; network pharmacology-based study was applied to probe the involved mechanisms; western blotting, immunocytochemistry, and luciferase reporter assay was used to further confirm the regulation of magnolol on JAK/STATs signaling pathway; surface plasmon resonance (SPR) assay and molecular docking were applied to manifest affinity with STAT3 and binding sites; overexpression of STAT3 was employed to verify whether magnolol attenuates IL-17A through STAT3 signaling pathway. ResultsIn vivo, magnolol alleviated loss of body weight and severity of EAE mice; magnolol improved lesions in spinal cords and attenuated CD45 infiltration, and serum cytokines levels; correspondingly, magnolol focused on inhibiting Th17 differentiation and IL-17A expression in splenocyte of EAE mice; moreover, magnolol selectively inhibited p-STAT3(Y705) and p-STAT4(Y693) of both CD4+ and CD8+ T cells in splenocyte of EAE mice. In vitro, magnolol selectively inhibited Th17 differentiation and IL-17A expression without impact on Treg cells; network pharmacology-based study revealed that magnolol perhaps diminished Th17 cell differentiation through regulating STAT family members; western blotting further confirmed that magnolol inhibited p-JAK2(Y1007) and selectively antagonized p-STAT3(Y705) and slightly decreased p-STAT4(Y693); magnolol antagonized both STAT3 nucleus location and transcription activity; magnolol had a high affinity with STAT3 and the specific binding site perhaps to be at SH2 domain; overexpression of STAT3 resulted in failed inhibition of magnolol on IL-17A. ConclusionMagnolol selectively inhibited Th17 differentiation and cytokine expression through selectively blocking of STAT3 resulting in decreased the ratio of Th17/Treg cells for treating MS, suggesting that the potential of magnolol for treating MS as novel STAT3 inhibitor.

Exploration of T cell immune responses by expression of a dominant-negative SHP1 and SHP2.

SHP1 and SHP2 are SH2 domain-containing proteins which have inhibitory phosphatase activity when recruited to phosphorylated ITIMs and ITSMs on inhibitory immune receptors. Consequently, SHP1 and SHP2 are key proteins in the transmission of inhibitory signals within T cells, constituting an important point of convergence for diverse inhibitory receptors. Therefore, SHP1 and SHP2 inhibition may represent a strategy for preventing immunosuppression of T cells mediated by cancers hence improving immunotherapies directed against these malignancies. Both SHP1 and SHP2 contain dual SH2 domains responsible for localization to the endodomain of inhibitory receptors and a protein tyrosine phosphatase domain which dephosphorylates and thus inhibits key mediators of T cell activation. We explored the interaction of the isolated SH2 domains of SHP1 and SHP2 to inhibitory motifs from PD1 and identified strong binding of both SH2 domains from SHP2 and more moderate binding in the case of SHP1. We next explored whether a truncated form of SHP1/2 comprising only of SH2 domains (dSHP1/2) could act in a dominant negative fashion by preventing docking of the wild type proteins. When co-expressed with CARs we found that dSHP2 but not dSHP1 could alleviate immunosuppression mediated by PD1. We next explored the capacity of dSHP2 to bind with other inhibitory receptors and observed several potential interactions. In vivo we observed that the expression of PDL1 on tumor cells impaired the ability of CAR T cells to mediate tumor rejection and this effect was partially reversed by the co-expression of dSHP2 albeit at the cost of reduced CAR T cell proliferation. Modulation of SHP1 and SHP2 activity in engineered T cells through the expression of these truncated variants may enhance T cell activity and hence efficacy in the context of cancer immunotherapy.

LncASAP1-IT1 promotes hepatocellular carcinoma progression through the regulation of the miR-1294/TGFBR1 pathway in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the most common tumor with severe morbidity and high mortality. The lncRNA ASAP1-IT1 [the intronic transcript 1 (IT-1) of ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1)] have been shown to promote tumor formation in a variety of cancers. This study sought to investigate the effects of dysregulated ASAP1-IT1 on the biological processes of HCC. The expression levels of ASAP1-IT1 in 30 paired HCC and adjacent non-tumor tissues were measured by real-time-quantitative polymerase chain reaction (RT-qPCR). Several functional tests were performed to investigate the molecular mechanism of ASAP1-IT1 in HCC progression. Our study showed that ASAP1-IT1 was highly expressed in the HCC tissues and cell lines. The knockdown of ASAP1-IT1 inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progression and enhanced the sorafenib sensitivity of the HCC cells. Further investigations revealed that ASAP1-IT1 served as a sponge of microRNA-1294 (miR-1294) to promote transforming growth factor beta receptor 1 (TGFBR1) expression. In addition, the tumor-promoting effect of ASAP1-IT1 was blocked by inhibiting miR-1294/TGFBR1. Tumorigenic assays in nude mice demonstrated that the inhibition of ASAP1-IT1 inhibited the growth of HCC in vivo. These results suggest that lncASAP1-IT1 promotes HCC development by targeting TGFBR1 through miR-1294, which provides a potential target for HCC diagnosis and treatment.

Towards the development of a chemical probe targeting the disruption of SYK tandem SH2 domain and FCεR1γ interactions for Alzheimer’s disease

AbstractBackgroundSpleen tyrosine kinase (SYK) and FCεR1γ were identified as targets for Alzheimer’s disease (AD) via multi‐omics human data by AMP‐AD teams. These proteins were selected for further target validation and medicinal chemistry efforts by members of the Target Enablement to Accelerate Therapy Development for Alzheimer’s Disease (TREAT‐AD) program. SYK has a reported role in AD (PMID: 31324720), and SYK activation can occur upon binding to the phospho‐ITAM region of the FCεR1γ receptor via the tandem SH2 domains (tSH2) of SYK (PMID: 29440271). Therefore, disruption of this interaction is proposed to slow AD related pathology. Through the generation of target enabling packages (TEPs) we aim to provide experimental validation of these targets for AD and develop a high‐quality chemical probe that targets this interaction.MethodFirstly, tSH2 domains of SYK were purified, followed by crystallography of these tSH2 in the presence of phospho‐ITAM‐containing peptides. A TR‐FRET‐based assay was developed using the SYK tSH2 domain and phospho‐ITAM peptide of FCεR1γ. This assay was miniaturized for ultra‐high‐throughput screening (uHTS) of a screening library of compounds. In parallel, a DNA encoded library (DEL) screen was executed to identify compounds that bind the SYK tSH2 domain. The hits generated using these hit finding campaigns were validated in dose‐response and via orthogonal biophysical methods. Medicinal chemistry campaigns to expand upon these hits are underway.ResultWe have crystallography data of the SYK tSH2 domains bound to phospho‐ITAM peptides and have established a TR‐FRET based assay to identify inhibitors of the interaction between FCεR1γ and SYK. Hit compounds have been identified through uHTS and DEL screening methods. Further validation of these hits in orthogonal assays are underway, alongside medicinal chemistry efforts to identify more potent analogues.ConclusionThrough the generation of a SYK/FCεR1γ TEP, we have provided the tools to allow for experimental validation of these target for AD and have begun to generate inhibitors of this interaction.

Construction of T cell exhaustion model for predicting survival and immunotherapy effect of bladder cancer based on WGCNA.

The prognosis of bladder cancer (BLCA) and response to immune checkpoint inhibitors (ICIs) are determined by multiple factors. Existed biomarkers for predicting the effect of immunotherapy cannot accurately predict the response of BLCA patients to ICIs. To further accurately stratify patients' response to ICIs and identify potential novel predictive biomarkers, we used the known T cell exhaustion (TEX)-related specific pathways, including tumor necrosis factor (TNF), interleukin (IL)-2, interferon (IFN)-g, and T- cell cytotoxicpathways, combined with weighted correlation network analysis (WGCNA) to analyze the characteristics of TEX in BLCA in detail, constructed a TEX model. This model including 28 genes can robustly predict the survival of BLCA and immunotherapeutic efficacy. This model could divide BLCA into two groups, TEXhigh and TEXlow, with significantly different prognoses, clinical features, and reactivity to ICIs. The critical characteristic genes, such as potential biomarkers Charged Multivesicular Body Protein 4C (CHMP4C), SH2 Domain Containing 2A (SH2D2A), Prickle Planar Cell Polarity Protein 3 (PRICKLE3) and Zinc Finger Protein 165 (ZNF165) were verified in BLCA clinical samples by real-time quantitative chain reaction (qPCR) and immunohistochemistry (IHC). Our findings show that the TEX model can serve as biological markers for predicting the response to ICIs, and the involving molecules in the TEX model might provide new potential targets for immunotherapy in BLCA.

Postsynaptic Protein Shank3a Deficiency Synergizes with Alzheimer's Disease Neuropathology to Impair Cognitive Performance in the 3xTg-AD Murine Model.

Synaptic loss is intrinsically linked to Alzheimer's disease (AD) neuropathology and symptoms, but its direct impact on clinical symptoms remains elusive. The postsynaptic protein Shank3 (SH3 and multiple ankyrin repeat domains) is of particular interest, as the loss of a single allele of the SHANK3 gene is sufficient to cause profound cognitive symptoms in children. We thus sought to determine whether a SHANK3 deficiency could contribute to the emergence or worsening of AD symptoms and neuropathology. We first found a 30%-50% postmortem loss of SHANK3a associated with cognitive decline in the parietal cortex of individuals with AD. To further probe the role of SHANK3 in AD, we crossed male and female 3xTg-AD mice modelling Aβ and tau pathologies with Shank3a-deficient mice (Shank3Δex4-9). We observed synergistic deleterious effects of Shank3a deficiency and AD neuropathology on object recognition memory at 9, 12, and 18 months of age and on anxious behavior at 9 and 12 months of age in hemizygous Shank3Δex4-9-3xTg-AD mice. In addition to the expected 50% loss of Shank3a, levels of other synaptic proteins, such as PSD-95, drebrin, and homer1, remained unchanged in the parietotemporal cortex of hemizygous Shank3Δex4-9 animals. However, Shank3a deficiency increased the levels of soluble Aβ42 and human tau at 18 months of age compared with 3xTg-AD mice with normal Shank3 expression. The results of this study in human brain samples and in transgenic mice are consistent with the hypothesis that Shank3 deficiency makes a key contribution to cognitive impairment in AD.SIGNIFICANCE STATEMENT Although the loss of several synaptic proteins has been described in Alzheimer's disease (AD), it remains unclear whether their reduction contributes to clinical symptoms. The results of this study in human samples show lower levels of SHANK3a in AD brain, correlating with cognitive decline. Data gathered in a novel transgenic mouse suggest that Shank3a deficiency synergizes with AD neuropathology to induce cognitive impairment, consistent with a causal role in AD. Therefore, treatment aiming at preserving Shank3 in the aging brain may be beneficial to prevent AD.

Identification of SH2 domain-containing proteins and motifs prediction by a deep learning method

The Src Homology 2 (SH2) domain plays an important role in the signal transmission mechanism in organisms. It mediates the protein-protein interactions based on the combination between phosphotyrosine and motifs in SH2 domain. In this study, we designed a method to identify SH2 domain-containing proteins and non-SH2 domain-containing proteins through deep learning technology. Firstly, we collected SH2 and non-SH2 domain-containing protein sequences including multiple species. We built six deep learning models through DeepBIO after data preprocessing and compared their performance. Secondly, we selected the model with the strongest comprehensive ability to conduct training and test separately again, and analyze the results visually. It was found that 288-dimensional (288D) feature could effectively identify two types of proteins. Finally, motifs analysis discovered the specific motif YKIR and revealed its function in signal transduction. In summary, we successfully identified SH2 domain and non-SH2 domain proteins through deep learning method, and obtained 288D features that perform best. In addition, we found a new motif YKIR in SH2 domain, and analyzed its function which helps to further understand the signaling mechanisms within the organism.

Combined immunodeficiency caused by pathogenic variants in the ZAP70 C-terminal SH2 domain.

ZAP-70, a protein tyrosine kinase recruited to the T cell receptor (TCR), initiates a TCR signaling cascade upon antigen stimulation. Mutations in the ZAP70 gene cause a combined immunodeficiency characterized by low or absent CD8+ T cells and nonfunctional CD4+ T cells. Most deleterious missense ZAP70 mutations in patients are located in the kinase domain but the impact of mutations in the SH2 domains, regulating ZAP-70 recruitment to the TCR, are not well understood. Genetic analyses were performed on four patients with CD8 lymphopenia and a high resolution melting screening for ZAP70 mutations was developed. The impact of SH2 domain mutations was evaluated by biochemical and functional analyses as well as by protein modeling. Genetic characterization of an infant who presented with pneumocystis pneumonia, mycobacterial infection, and an absence of CD8 T cells revealed a novel homozygous mutation in the C-terminal SH2 domain (SH2-C) of the ZAP70 gene (c.C343T, p.R170C). A distantly related second patient was found to be compound heterozygous for the R170C variant and a 13bp deletion in the ZAP70 kinase domain. While the R170C mutant was highly expressed, there was an absence of TCR-induced proliferation, associated with significantly attenuated TCR-induced ZAP-70 phosphorylation and a lack of binding of ZAP-70 to TCR-ζ. Moreover, a homozygous ZAP-70 R192W variant was identified in 2 siblings with combined immunodeficiency and CD8 lymphopenia, confirming the pathogenicity of this mutation. Structural modeling of this region revealed the critical nature of the arginines at positions 170 and 192, in concert with R190, forming a binding pocket for the phosphorylated TCR-ζ chain. Deleterious mutations in the SH2-C domain result in attenuated ZAP-70 function and clinical manifestations of immunodeficiency.

The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis.

The multidomain adaptor protein amphiphysin-1 (Amph1) is an important coordinator of clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid-binding N-BAR (Bin/Amphiphysin/Rvs) domain, central proline-rich (PRD) and clathrin/AP2 (CLAP) domains, and a C-terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain-specific interactions of Amph1 were validated using in vitro GST pull-down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1-endophilin A1 complex is dependent on the phosphorylation status of Amph1-S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation-dependent Amph1-endophilin A1 interaction in efficient SV endocytosis.

Stress granule activation attenuates lipopolysaccharide-induced cardiomyocyte dysfunction

BackgroundSepsis is the leading cause of death in intensive care units. Sepsis-induced myocardial dysfunction, one of the most serious complications of sepsis, is associated with higher mortality rates. As the pathogenesis of sepsis-induced cardiomyopathy has not been fully elucidated, there is no specific therapeutic approach. Stress granules (SG) are cytoplasmic membrane-less compartments that form in response to cellular stress and play important roles in various cell signaling pathways. The role of SG in sepsis-induced myocardial dysfunction has not been determined. Therefore, this study aimed to determine the effects of SG activation in septic cardiomyocytes (CMs).MethodsNeonatal CMs were treated with lipopolysaccharide (LPS). SG activation was visualized by immunofluorescence staining to detect the co-localization of GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and T cell-restricted intracellular antigen 1 (TIA-1). Eukaryotic translation initiation factor alpha (eIF2α) phosphorylation, an indicator of SG formation, was assessed by western blotting. Tumor necrosis factor alpha (TNF-α) production was assessed by PCR and enzyme-linked immunosorbent assays. CMs function was evaluated by intracellular cyclic adenosine monophosphate (cAMP) levels in response to dobutamine. Pharmacological inhibition (ISRIB), a G3BP1 CRISPR activation plasmid, and a G3BP1 KO plasmid were employed to modulate SG activation. The fluorescence intensity of JC-1 was used to evaluate mitochondrial membrane potential.ResultsLPS challenge in CMs induced SG activation and resulted in eIF2α phosphorylation, increased TNF-α production, and decreased intracellular cAMP in response to dobutamine. The pharmacological inhibition of SG (ISRIB) increased TNF-α expression and decreased intracellular cAMP levels in CMs treated with LPS. The overexpression of G3BP1 increased SG activation, attenuated the LPS-induced increase in TNF-α expression, and improved CMs contractility (as evidenced by increased intracellular cAMP). Furthermore, SG prevented LPS-induced mitochondrial membrane potential dissipation in CMs.ConclusionSG formation plays a protective role in CMs function in sepsis and is a candidate therapeutic target.

STS-1 and STS-2, Multi-Enzyme Proteins Equipped to Mediate Protein-Protein Interactions.

STS-1 and STS-2 form a small family of proteins that are involved in the regulation of signal transduction by protein-tyrosine kinases. Both proteins are composed of a UBA domain, an esterase domain, an SH3 domain, and a PGM domain. They use their UBA and SH3 domains to modify or rearrange protein-protein interactions and their PGM domain to catalyze protein-tyrosine dephosphorylation. In this manuscript, we discuss the various proteins that have been found to interact with STS-1 or STS-2 and describe the experiments used to uncover their interactions.