Signal Transducing Adaptor Molecule Research Articles

Deciphering the cleavage sites of 3C-like protease in Gammacoronaviruses and Deltacoronaviruses

Coronaviruses replicate by using the 3C-like protease (3CLpro) to cleave polyprotein precursors and host proteins. However, current tools for identifying 3CLpro cleavage sites are limited, particularly in Gammacoronaviruses (GammaCoV) and Deltacoronaviruses (DeltaCoV). This study aims to fill this gap by identifying 3CLpro cleavage sites in these viruses to provide deeper insights into their pathogenic mechanisms. By integrating sequence alignments and structural model comparisons, we developed a position-specific scoring matrix (PSSM) based on self-cleavage motifs, revealing specific preferences for each residue. Utilizing AlphaFold2's predicted alignment error (PAE) and predicted local distance difference test (pLDDT), we found that most cleavage sequences are located in regions with high PAE and low pLDDT values. KEGG pathway analysis showed that potential host protein cleavage targets are mainly concentrated in pathways related to nucleo-cytoplasmic transport and endocytosis. Through in vitro cleavage experiments and mutational analysis, we identified and validated three high-scoring proteins—nucleoporin 58 (NUP58), cell division cycle 73 (CDC73), and signal transducing adaptor molecule 2 (STAM2). These findings suggest that 3CLpro not only plays a vital role in viral replication but may also influence host cell functions by cleaving host proteins. This study provides an effective tool for identifying 3CLpro cleavage sites, revealing the pathogenic mechanisms of coronaviruses, and offering new insights for developing potential therapeutic targets.

Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein–protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.

The role of RNA viruses in human cancers

Many RNA viruses have been reported to be oncogenic (or carcinogenic) in a variety of animal and human cancers. The increase in the incidence and prevalence of cancer-causing viruses in human populations can be known as a key precursor to the development of various cancers. The retrovirus family and Hepatitis C virus (HCV) are also reported to cause cancer. Viral oncoproteins such as Tax of HTLV 1 interacts with cellular ubiquitination complex such as cyclindromatosis tumor suppressor, ubiquitin-specific proteases 7, 11, 15 and 20, A-20 and signal-transducing adaptor molecule binding protein-like-1 in order to improve the cellular signaling pathways. The viral oncoproteins binding to DUB, leading to proliferation of virus-infected cells and cell transformation. Proto-oncogenes (c-onc genes) are the cellular form of v-onc genes. The activation of c-onc genes leads to cell growth. C-onc genes are transformed into an oncogenic form by viral infection. C-onc genes play some roles such as protein kinases, growth factors, growth factor receptors, and DNA binding proteins. The study of transforming retroviruses and their oncogenes and the multiple mechanisms deployed by other RNA viruses to use the growth-suppressive and proapoptotic function of tumor suppressor genes has been added to our current understanding of cancer biology. Oncogenic RNA viruses are important experimental models to study molecular investigation such as cellular networks, including the discovery of oncogenes and tumor suppressors. Understanding of different strategies of RNA viruses as well as the function of their proteins helps to make more extensive plans regarding the adoption of follow-up, prevention and treatment strategies in cancer patients caused by viral origin.

STAM2 negatively regulates the MyD88-mediated NF-κB signaling pathway in miiuy croaker, Miichthys miiuy

Signal transducing adapter molecule 2 (STAM2), a member of the Signal Transducing Adapter Molecule (STAM) family, is a protein with significant implications in diverse signaling pathways and endocytic membrane trafficking. However, the role of the STAM2, especially in fish, remains largely unknown. In this study, we discovered that STAM2 negatively regulates the NF-κB signaling pathway, and its inhibitory effect is enhanced upon LPS induction. Our study confirmed that STAM2 can enhance the degradation of myeloid differentiation primary-response protein 88 (MyD88), an upstream regulator of NF-κB pathway. Furthermore, the UIM domain of STAM2 is important for the inhibition of MyD88. Mechanistically, STAM2 inhibits the NF-κB signaling pathway by targeting the MyD88 autophagy pathway. In addition, we showed that STAM2 promotes the proliferation of Vibrio harveyi. In summary, our study reveals that STAM2 inhibits NF-κB signaling activation and mediates innate immunity in teleost via the autophagy pathway.

Analysis of the neuromuscular deficits caused by STAM1 deficiency

The endosomal sorting complexes required for transport (ESCRT) pathway is composed of a series of protein complexes that are essential for sorting cargo through the endosome. In neurons, the ESCRT pathway is a key mediator of many cellular pathways that regulate neuronal morphogenesis as well as synaptic growth and function. The ESCRT-0 complex, consisting of HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), acts as a gate keeper to this pathway, ultimately determining the fate of the endosomal cargo. We previously showed that a single nucleotide substitution in Hgs results in structural and functional changes in the nervous system of teetering mice. To determine if these changes occurred as a function of HGS's role in the ESCRT pathway and its association with STAM1, we investigated if STAM1 deficiency also leads to a similar impairment of the nervous system. In contrast to teetering mice that die within 5 weeks of age and exhibit reduced body mass, 1-month-old Stam1 knockout mice were not visibly different from controls. However, by 3 months of age, STAM1 deficiency caused reduced muscle mass, strength, and motor performance. These changes in motor function did not correlate with either a loss in motor neuron number or abnormal myelination of peripheral nerves. Instead, the motor endplate structure was altered in the Stam1 knockout mice by 1 month of age and continued to degenerate over time, correlating with a significant reduction in muscle fiber size and increased expression of the embryonic γ acetylcholine receptor (AChR) subunit at 3 months of age. There was also a significant reduction in the levels of two presynaptic SNARE proteins, VTI1A and VAMP2, in the motor neurons of the Stam1 knockout mice. As loss of STAM1 expression replicates many of the structural changes at the motor endplates that we have previously reported with loss of HGS, these results suggest that the HGS/STAM1 complex plays a critical role in maintaining synaptic structure and function in the mammalian nervous system.

β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination

G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to βarrestins. In addition, βarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an invitro reconstituted system and show that βarrestin1 (βarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that βarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the βarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.

P38 Integrating proteomics and genomics to investigate the mechanisms of paradoxical eczema developing in patients with psoriasis treated with biologics: a UK multicentre, longitudinal, cohort study

Abstract Few studies have explored the immunology and genetic risk of paradoxical eczema occurring in patients with psoriasis treated with biologics. This study aims to describe the systemic inflammatory signature of paradoxical eczema using proteomics and explore whether it is genetically mediated. Participants were adults treated with biologics for plaque psoriasis, who were corecruited to the British Association of Dermatologists Biologics and Immunomodulators Register (BADBIR) and Biomarkers of Systemic Treatment Outcomes in Psoriasis (BSTOP) studies. To identify differentially expressed proteins and enriched gene sets, we used the Olink Target 96 Inflammation panel on 256 serum samples from BSTOP for 71 cases with paradoxical eczema and 75 controls. Paradoxical eczema was defined as eczema, atopic eczema or atopic dermatitis occurring during biologic exposure. Case samples across three time points [T1: prebiologic (n = 42); T2, postbiologic (n = 45); T3, postparadoxical eczema (n = 41)] were matched 1 : 1 with control samples. Case samples at T2 and T3 were taken during exposure to the same biologic that caused paradoxical eczema. Genes contributing to proteomic signatures were selected, and single-nucleotide polymorphisms (SNPs) that were nonsynonymous or associated with significant expression quantitative trait loci were used to create polygenic risk scores (PRS) in a genotyped cohort of 88 paradoxical eczema cases and 3124 psoriasis controls. Signal transducing adapter molecule 1-binding protein expression was lower in cases at T1 than in controls (log-fold change −0.44, adjusted P = 0.022); no other proteins achieved statistical significance at equivalent time points. Eleven gene sets, including cytokine and chemokine pathways, were enriched in cases at T2 and 10 at T3. Of the 39 proteins contributing to enriched gene sets, 21 are elevated in the sera of patients with atopic dermatitis. A PRS including 38 SNPs linked to enriched gene sets was associated with paradoxical eczema (adjusted P = 0.046). We subdivided the SNPs into those linked to specific gene sets, such as chemokine, cytokine and interleukin (IL)-10 signalling, to generate 10 partitioned PRS. Seven were associated with paradoxical eczema (adjusted P < 0.05). These SNPs do not map to known atopic loci. The paradoxical eczema systemic inflammatory proteome trends toward atopic dermatitis at a gene set level and is detectable before the onset of the phenotype. This signature may be mediated genetically by SNPs associated with these gene sets. Further studies are needed to explore the role of specific pathways (such as IL-10 signalling) in paradoxical eczema and define the inflammatory profile in the skin.

STAM transports STING oligomers into extracellular vesicles, down-regulating the innate immune response.

Stimulator of interferon genes (STING) mediates the innate immune response against damaged endogenous double-strand DNA and exogenous virus infection. The location of STING is critical to the accurate control of defence signalling pathways. Recently, the effects of extracellular vesicles (EVs) in the regulation of innate immune signalling have been reported. Nevertheless, the particular roles played by STING in EVs and the related mechanisms have remained largely unknown. Herein, we report that when STING was activated in cells, EVs derived from these cells carried STING oligomers. Signal transducing adapter molecule 1 (STAM) was found to be a STING transporter that directly interacted with STING and facilitated STING transport into EVs. Importantly, the translocation of STING into EVs was a mechanism by which STING was degraded, suppressing the innate immune response. In summary, we elucidated the mechanism and function of the translocation of STING into EVs, adding to the understanding of STING activity regulation.

G protein–coupled receptor kinase phosphorylation of distal C-tail sites specifies βarrestin1-mediated signaling by chemokine receptor CXCR4

G protein–coupled receptor (GPCR) kinases (GRKs) and arrestins mediate GPCR desensitization, internalization, and signaling. The spatial pattern of GPCR phosphorylation is predicted to trigger these discrete GRK and arrestin-mediated functions. Here, we provide evidence that distal carboxyl-terminal tail (C-tail), but not proximal, phosphorylation of the chemokine receptor CXCR4 specifies βarrestin1 (βarr1)-dependent signaling. We demonstrate by pharmacologic inhibition of GRK2/3-mediated phosphorylation of the chemokine receptor CXCR4 coupled with site-directed mutagenesis and bioluminescence resonance energy transfer approaches that distal, not proximal, C-tail phosphorylation sites are required for recruitment of the adaptor protein STAM1 (signal-transducing adaptor molecule) to βarr1 and focal adhesion kinase phosphorylation but not extracellular signal–regulated kinase 1/2 phosphorylation. In addition, we show that GPCRs that have similarly positioned C-tail phosphoresidues are also able to recruit STAM1 to βarr1. However, although necessary for some GPCRs, we found that distal C-tail sites might not be sufficient to specify recruitment of STAM1 to βarr1 for other GPCRs. In conclusion, this study provides evidence that distal C-tail phosphorylation sites specify GRK–βarrestin-mediated signaling by CXCR4 and other GPCRs.

Carbon Nanotube Exposure Triggers a Cerebral Peptidomic Response: Barrier Compromise, Neuroinflammation, and a Hyperexcited State

The unique physicochemical properties of carbon nanomaterials and their ever-growing utilization generate a serious concern for occupational risk. Pulmonary exposure to these nanoparticles induces local and systemic inflammation, cardiovascular dysfunction, and even cognitive deficits. While multiple routes of extrapulmonary toxicity have been proposed, the mechanism for and manner of neurologic effects remain minimally understood. Here, we examine the cerebral spinal fluid (CSF)-derived peptidomic fraction as a reflection of neuropathological alterations induced by pulmonary carbon nanomaterial exposure. Male C57BL/6 mice were exposed to 10 or 40 µg of multi-walled carbon nanotubes (MWCNT) by oropharyngeal aspiration. Serum and cerebrospinal fluids were collected 4 h post-exposure. An enriched peptide fraction of both biofluids was analyzed using ion mobility-enabled data-independent mass spectrometry for label-free quantification. MWCNT exposure induced a prominent peptidomic response in the blood and CSF; however, correlation between fluids was limited. Instead, we determined that a MWCNT-induced peptidomic shift occurred specific to the CSF with 292 significant responses found that were not in serum. Identified MWCNT-responsive peptides depicted a mechanism involving aberrant fibrinolysis (fibrinopeptide A), blood-brain barrier permeation (homeobox protein A4), neuroinflammation (transmembrane protein 131 L) with reactivity by astrocytes and microglia, and a pro-degradative (signal transducing adapter molecule, phosphoglycerate kinase), anti-plastic (AF4/FMR2 family member 1, vacuolar protein sorting-associated protein 18) state with the excitation-inhibition balance shifted to a hyperexcited (microtubule-associated protein 1B) phenotype. Overall, the significant pathologic changes observed were consistent with early neurodegenerative disease and were diagnostically reflected in the CSF peptidome. Impact Statement These studies demonstrate in a model of nanotube pulmonary exposure that there is a distinct CSF peptidomic response diagnostic of a compromised blood-brain barrier, neuroinflammation, and a hyperexcited neuronal state that is consistent with early-pathobiology of neurodegenerative disease.

STAM Prolongs Clear Cell Renal Cell Carcinoma Patients' Survival via Inhibiting Cell Growth and Invasion.

Background: Signal transducing adaptor molecule 1 (STAM1) was considered to mediate cell growth and be involved in multiple signaling pathways; however, no research on the role of STAM1 in any tumors has been published yet. Our study aimed to investigate the prognostic value of STAM1 for clear cell renal cell carcinoma (ccRCC) and its role in modulating cancer cell function.Methods: Data from The Cancer Genome Atlas (TCGA) in December 2019 were used to examine the role of STAM1 in indicating ccRCC patients' survival. A purchased tissue microarray (TM) and fresh ccRCC renal tissues were used for further validation. Then, STAM1 was overexpressed in human ccRCC cell lines for in vitro assays. Finally, bioinformatics was performed for STAM1 protein–protein interaction (PPI) network construction and functional analyses.Results: A total of 539 ccRCC and 72 control samples were included for the TCGA cohort, and 149 ccRCC and 29 control slices were included for the TM cohort. In the TCGA and TM cohorts, we found that STAM1 expression was lower in ccRCC compared with normal adjacent non-cancerous renal tissues (P < 0.0001 for both cohorts). STAM1 downregulation was also related to significantly shorter overall survival (OS) (P < 0.0001 for both cohorts). In the TCGA cohort, reduced STAM1 expression was also associated with aggressive features of the tumor. Under multivariate analyses, STAM1 was demonstrated to be an independent prognostic factor for ccRCC survival in both TCGA (HR = 0.52, 95% CI: 0.33–0.84, P = 0.007) and TM cohorts (HR = 0.12, 95% CI: 0.04–0.32, P < 0.001). Our in vitro experiments showed that STAM1 inhibited cell viability, invasion, and migration in ccRCC cell lines. In PPI network, 10 candidate genes categorized into five biological processes were found to be closely related to STAM1.Conclusion: STAM1 is a promising prognostic biomarker for predicting ccRCC survival outcomes. Preliminary pathogenesis is demonstrated by our in vitro experiments. Further pathological mechanisms of STAM1 in modulating ccRCC require comprehensive laboratory and clinical studies.

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

A non–GPCR-binding partner interacts with a novel surface on β-arrestin1 to mediate GPCR signaling

The multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer-based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin-dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling.

Exploring Potential Proteomic Biomarkers for Prognosis of Infective Endocarditis through Profiled Autoantibodies by an Immunomics Protein Array Technique.

Though infective endocarditis (IE) is a life-threatening cardiac infection with a high mortality rate, the effective diagnostic and prognostic biomarkers for IE are still lacking. The aim of this study was to explore the potential applicable proteomic biomarkers for IE through the Immunome™ Protein Array system. The system was employed to profile those autoantibodies in IE patients and control subjects. Our results showed that interleukin-1 alpha (IL1A), nucleolar protein 4 (NOL4), tudor and KH domain-containing protein (TDRKH), G antigen 2B/2C (GAGE2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and X antigen family member 2 (XAGE2) are highly differentially-expressed among IE and non-IE control. Furthermore, bactericidal permeability-increasing protein (BPI), drebrin-like protein (DBNL), signal transducing adapter molecule 2 (STAM2), cyclin-dependent kinase 16 (CDK16), BAG family molecular chaperone regulator 4 (BAG4), and nuclear receptor-interacting protein 3 (NRIP3) are differentially-expressed among IE and healthy controls. On the other hand, those previously identified biomarkers for IE, including erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, procalcitonin, and N-terminal-pro-B-type natriuretic peptide demonstrated only minor significance. With scientific rationalities for those highly differentially-expressed proteins, they could serve as potential candidates for diagnostic biomarkers of IE for further analysis.

Identifying β‐arrestin1 conformational states by a non‐GPCR binding partner

The G protein‐coupled receptor (GPCR) C‐X‐C motif chemokine receptor 4 (CXCR4), and its cognate chemokine CXCL12, play essential roles in hematopoiesis, cardiogenesis, neurogenesis and immune responses. In addition, CXCR4 signaling contributes to several aspects of cancer progression and other diseases. Despite its importance CXCR4 signaling remains poorly understood. Because of the critical involvement in human cancers, a comprehensive understanding of CXCR4 signaling is of great therapeutic significance. The present study is focused on a novel CXCR4 signaling pathway thatregulates chemotaxis via the adaptor protein β‐arrestin1 (βarr1). β‐arrestins typically bind to activated GPCRs to negatively regulate signaling by promoting desensitization and internalization, but β‐arrestins also positively promote signaling by interacting with non‐GPCR binding partners. It is relatively well known how β‐arrestins interact with activated GPCRs to promote their internalization and desensitization, however very little is known how β‐arrestins interact with non‐GPCR binding partners to initiate signaling. Previously, our lab showed that βarr1 interacts directly with the adaptor protein STAM1 (signal transducing adaptor molecule) to promote CXCR4‐dependent activation of focal adhesion kinase (FAK), however the mechanism remains unknown. Here, we tested the hypothesis that STAM1 induces a unique conformational signature in βarr1 that facilitates activation of FAK. To test this, we employed Site‐Directed Spin Labeling (SDSL) Electron Paramagnetic Resonance (EPR) spectroscopy. We employed double electron‐electron resonance (DEER) spectroscopy to determine if STAM1 induces a unique conformational signature on βarr1 by incorporating spin labels on selected double cysteine mutants of βarr1 and measuring distances between spin labeled residues upon STAM1 or GPCR binding. STAM1 binding induced similar conformational changes on βarr1 in one double cysteine labeled mutant, but not on two others, as compared to a GPCR. These data suggest that βarr1 undergoes a unique conformational change upon STAM1 binding, in contrast to the well‐established conformational change that occurs upon GPCR binding. To determine if this could be explained by the fact that STAM1 and GPCRs bind to different sites on βarr1 we employed CW(continuous wave)‐EPR spectroscopy to map the STAM1 binding surface on βarr1 by monitoring spectral line‐shape changes of selected reporter sites in βarr1 in the presence or absence of STAM1. Spin labels attached to selected sites on the front surface of βarr1 did not show any side chain immobilization when bound to STAM1, suggesting that STAM1 does not bind to the front surface. In contrast, we identified two residues located on the back surface of βarr1 that showed significant spin label immobilization when bound to STAM1, suggesting STAM1 binds to this surface of βarr1. As GPCRs and other non‐GPCR binding partners typically bind to the front surface of β‐arrestins, our data suggest that STAM1 binds to the back surface on βarr1, which is likely linked to discrete βarr1 conformational dynamics. We propose that STAM1 induces a unique βarr1 conformational signature, which is required for CXCR4‐dependent activation of FAK and chemotaxis.Support or Funding InformationGM106727 06A1

The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network.

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

Effect of Acute Exercise on Skeletal Muscle Exosome Biogenesis

Exercise training promotes a wide range of beneficial adaptations. Skeletal muscle is now considered an endocrine organ. Exosomes, small microvesicles, are produced by and participate in the endocrine function of skeletal muscle. Exosome biogenesis is regulated in part by components of the multivesicular body (MVB) processing pathway: hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), signal transducing adapter molecule 1 (STAM), VTA1 homolog (VTA1), and vacuolar protein sorting-associated protein 4A (VPS4a). PURPOSE: Determine if exercise induces skeletal muscle exosome biogenesis. METHODS: Twelve lean, young men completed acute aerobic cycling at 55% VO2Max for 45 minutes followed immediately by single leg knee extensor resistance exercise (3 sets, 8-12 reps, 55% 1-RM). Vastus lateralis biopsies were obtained prior to (PRE) and 1-hour post aerobic (AEX) and aerobic+resistance (A+REX) exercise. Gene expression for proteins in the MVB pathway was analyzed using rt- PCR. RESULTS: There was no effect of exercise on STAM (PRE: 1.0; AEX: 1.03; A+REX: 1.28, AU), VTA1 (PRE: 1.0; AEX: 0.93; A+REX: 1.14, AU), or VPS4a (PRE: 1.0; AEX: 0.93; A+REX: 1.06, AU) . There was a trend for an increase in HGS at 1 hr post-exercise (PRE: 1.0; AEX: 0.97; A+REX: 1.27, AU). CONCLUSIONS: There was no effect of acute exercise on the gene expression of components of the exosome biogenesis pathway. However, activation of exosome biogenesis may be evident at different time points post exercise or with greater exercise intensities.

β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 and its chemokine ligand CXCL12 mediate directed cell migration during organogenesis, immune responses, and metastatic disease. However, the mechanisms governing CXCL12/CXCR4-dependent chemotaxis remain poorly understood. Here, we show that the β-arrestin1·signal-transducing adaptor molecule 1 (STAM1) complex, initially identified to govern lysosomal trafficking of CXCR4, also mediates CXCR4-dependent chemotaxis. Expression of minigene fragments from β-arrestin1 or STAM1, known to disrupt the β-arrestin1·STAM1 complex, and RNAi against β-arrestin1 or STAM1, attenuates CXCL12-induced chemotaxis. The β-arrestin1·STAM1 complex is necessary for promoting autophosphorylation of focal adhesion kinase (FAK). FAK is necessary for CXCL12-induced chemotaxis and associates with and localizes with β-arrestin1 and STAM1 in a CXCL12-dependent manner. Our data reveal previously unknown roles in CXCR4-dependent chemotaxis for β-arrestin1 and STAM1, which we propose act in concert to regulate FAK signaling. The β-arrestin1·STAM1 complex is a promising target for blocking CXCR4-promoted FAK autophosphorylation and chemotaxis.

NMR Reveals the Interplay among the AMSH SH3 Binding Motif, STAM2, and Lys63-Linked Diubiquitin

AMSH [associated molecule with a Src homology 3 domain of signal transducing adaptor molecule (STAM)] is one of the deubiquitinating enzymes associated in the regulation of endocytic cargo trafficking. It shows an exquisite selectivity for Lys63-linked polyubiquitin chains that are the main chains involved in cargo sorting. The first step requires the ESCRT-0 complex that comprises the STAM and hepatocyte growth factor-regulated substrate (Hrs) proteins. Previous studies have shown that the presence of the STAM protein increases the efficiency of Lys63-linked polyubiquitin chain cleavage by AMSH, one of the deubiquitinating enzyme involved in lysosomal degradation. In the present study, we are seeking to understand if a particular structural organization among these three key players is responsible for the stimulation of the catalytic activity of AMSH. To address this question, we first monitored the interaction between the ubiquitin interacting motif (UIM)-SH3 construct of STAM2 and the Lys63-linked diubiquitin (Lys63-Ub2) chains by means of NMR. We show that Lys63-Ub2 is able to bind either the UIM or the SH3 domain without any selectivity. We further demonstrate that the SH3 binding motif (SBM) of AMSH (AMSH-SBM) outcompetes Lys63-Ub2 for binding SH3. Additionally, we show how different AMSH-SBM variants, modified by their sequence and length, exhibit similar equilibrium dissociation constants when binding SH3 but significantly differ in their dissociation rate constants. Finally, we report the solution NMR structure of the AMSH-SBM/SH3 complex and propose a structural organization where the AMSH-SBM interacts with the STAM2-SH3 domain and contributes to the correct positioning of AMSH prior to polyubiquitin chains' cleavage.

Protective effects of elafin against adult asthma

Elafin inhibits serine proteases, such as human neutrophil elastase and proteinase 3, to prevent excessive damage during inflammation. However, the relationship between elafin and asthma is still unclear. Microarray technology was used to evaluate smoking- and asthma-related biomarkers in a discovery-driven manner. We identified candidate genes, e.g., proteinase inhibitor 3 (PI3), related to asthma and smoking from gene expression microarray data sets and evaluated their potential as biomarkers for asthma. We used human genome microarray data sets from smoking- and asthma-related gene expression data sets and performed real-time quantitative polymerase chain reaction to measure and validate differences in gene expression. We also recruited adult patients with asthma and age- and sex-matched control patients who were administered a structured questionnaire and evaluated for lung function and plasma elafin levels, which are encoded by the PI3 gene. Six significantly altered candidate genes, PI3, protein kinase C iota, phosphoserine phosphatase, IQ motif-containing GTPase activating protein 1, interleukin 13 receptor α 1, and signal transducing adaptor molecule SH3 domain and ITAM motif 2, were identified from comparisons across the four asthma- and four smoking-related data sets included in this study. An in vitro study of human airway epithelial cells (A549) and a human monocytic cell line (THP-1) demonstrated that PI3 messenger RNA levels were significantly altered by nicotine exposure. Elafin concentration was significantly higher in control patients than in patients with asthma (p < 0.001). The plasma elafin concentration in the highest quartile (≥12.69 ng/mL) was inversely associated with asthma (adjusted odds ratio 0.122 [95% confidence interval, 0.053-0.278]) compared with the lowest quartile (<5.82 ng/mL) after adjusting for age, sex, smoking status, waist-to-hip ratio, percentage predicted forced expiratory volume in 1 second, cockroaches in the home, incense burning, and family history. Our study revealed that high elafin levels identified in smoking- and asthma-related microarray data sets and an epidemiologic study significantly reduced the risk of asthma. Further studies of elafin as a potential therapy for asthma are warranted.