Adaptor Protein Research Articles

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions.

The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.

Bioinformatics Analysis of Hub Genes in Craniofacial Microsomia Combined With Congenital Heart Disease

Objective: The primary objective of this study was to investigate potential mechanisms and explore hub genes of craniofacial microsomia (CFM) patients associated with congenital heart defects (CHD). Methods: Initially, the authors acquired target gene data related to CFM and congenital cardiac anomalies. Subsequently, the authors established a protein-protein interaction (PPI) network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and molecular complex detection were conducted using Metascape. Finally, the authors hub genes were screened by the cytoHubba plugin. Results: A total of 43 CFM genes and 120 optimal CHD candidate genes were selected. The PPI networks for pathogenic genes contained 163 nodes and 1179 edges. Functional enrichment analysis largely focused on tissue formation and development. Five modules were identified from the PPI network, and 7 hub genes were screened out. The genes most relevant to CFM associated with congenital cardiac anomalies pathogenesis included fibroblast growth factor 3, GATA binding protein 3, nuclear factor of activated T cells 1, histone cell cycle regulator, EPAS1, mitogen-activated protein kinase 1, and CRK like proto-oncogene, adaptor protein. Conclusions: This study identified some significant hub genes, pathways, and modules of CFM associated with CHD by bioinformatics analyses. Our findings indicate that gene subfamilies fibroblast growth factor 3, GATA binding protein 3, nuclear factor of activated T cells 1, histone cell cycle regulator, EPAS1, mitogen-activated protein kinase 1, and CRK like proto-oncogene, adaptor protein may have had significant involvement in both CFM and CHD.

LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.

Analysis of the presence of anti-viral innate immune pathways in the Australian Haliotis laevigata

Our understanding of the specificities of innate immune protection against viral pathogens in non-model invertebrate species is limited, and comparative immunological approaches may offer opportunities to protect aquaculturally significant species such as abalone, against viral threats. Haliotid herpesvirus (HaHV-1) is a re-emerging viral pathogen of abalone, and has a high mortality rate in the dominant wild and farmed species in Australia, the Australian greenlip abalone (Haliotis laevigata), the blacklip (Haliotis rubra) and a hybrid of the two. To gain a better understanding of the anti-viral immune system counterparts of Australian abalone, gene mining strategies and domain analysis of the recently released H. laevigata draft genome was performed, with comparisons made to the better studied mollusc, the oyster (Crassostrea gigas). Significant divergence in members of the TLR, cytosolic RNA/DNA and RNA interference signalling pathways was observed between the two molluscs, with the oyster more closely reflecting that of the mammalian immune response. Interestingly, a homolog for the anti-viral RNA sensing adaptor protein Mitochondrial anti-viral-signalling protein was absent in H. laevigata, as well as the 4 other abalone protein databases currently available, despite the presence of a RIG-I homolog. Additionally, STING, a member of the dsDNA sensing pathway was absent in H. laevigata, as well as 2 other abalone species, however is present in the oyster genome, and two additional abalone species (Haliotis rufescens, Haliotis discus hannai).This work provides a better understanding of the key features of the abalone anti-viral innate immune signalling system and may provide much needed information towards the development of anti-viral strategies in these animals.

Phospho-mimetic CD3ε variants prevent TCR and CAR signaling

IntroductionAntigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome.MethodsWe mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM.ResultsIntegrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK.ConclusionsThe data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CAR signaling and highlights the key role of CD3ε signalosome to tune signal transduction.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, result in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation, and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of PCOS induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

Grape seed-derived procyanidins decreases neuropathic pain and nerve regeneration by suppression of TLR4-Myd88 signaling.

Recent studies have shown that peripheral nerve regeneration process is closely related to neuropathic pain. Toll-like receptor 4 (TLR4) signaling was involved in different types of pain and nerve regeneration. TLR4 induced the recruitment of myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB-depended transcriptional process in sensory neurons and glial cells, which produced multiple cytokines and promoted the induction and persistence of pain. Our study aimed to investigate procyanidins's effect on pain and nerve regeneration via TLR4-Myd88 signaling. Spinal nerve ligation (SNL) model was established to measure the analgesic effect of procyanidins. Anatomical measurement of peripheral nerve regeneration was measured by microscopy and growth associated protein 43 (GAP43) staining. Western blotting and/or immunofluorescent staining were utilized to detect TLR4, myeloid differentiation factor-88 adaptor protein (MyD88), ionized calcium-binding adapter molecule 1 (IBA1) and nuclear factor kappa-B-p65 (NF-κB-p65) expression, as well as the activation of astrocyte and microglia. The antagonist of TLR4 (LPS-RS-Ultra, LRU) were intrathecally administrated to assess the behavioral effects of blocking TLR4 signaling on pain and nerve regeneration Result:Procyanidins reduced mechanical allodynia, thermal hyperalgesia and significantly suppressed the number of nerve fibers regenerated and the degree of myelination in SNL model. Compared with sham group, TLR4, MyD88, IBA1 and phosphorylation of NF-κB-p65 were upregulated in SNL rats which were reversed by procyanidins administration. Additionally, procyanidins also suppressed activation of spinal astrocytes and glial cells. Suppression of TLR4-MyD88 signaling contributes to the alleviation of neuropathic pain and reduction of nerve regeneration by procyanidins.

Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique “internal-PDZ binding motif” (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.)

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Dab2 (Disabled-2), an adaptor protein, regulates self-renewal of hair follicle stem cells

Disabled 2 (Dab2), an adaptor protein, is up regulated in the hair follicle stem cells (HFSCs); however, its role in any tissue stem cells has not been studied. In the present study, we have reported that Dab2 conditional knockout (Dab2-cKO) mice exhibited a delay in the HF cycle due to perturbed activation of HFSCs. Further, Dab2-cKO mice showed a reduction in the number of HFSCs and reduced colony forming ability of HFSCs. Dab2-cKO mice showed extended quiescence of HFSCs concomitant with an increased expression of Nfatc1. Dab2-cKO mice showed a decreased expression of anti-aging genes such as Col17a1, decorin, Sirt2 and Sirt7. Dab2-cKO mice did not show full hair coat recovery in aged mice thereby suggesting an accelerated aging process. Overall, we unveil for the first time, the role of Dab2 that regulate activation and self-renewal of HFSCs.

Exposure to nanopollutants (nZnO) enhances the negative effects of hypoxia and delays recovery of the mussels’ immune system

Aquatic environments face escalating challenges from multiple stressors like hypoxia and nanoparticle exposure, with impact of these combined stressors on mussel immunity being poorly understood. We investigated the individual and combined effects of short-term and long-term hypoxia and exposure to zinc oxide nanoparticles (nZnO) on immune system of the mussels (Mytilus edulis). Hemocyte functional traits (mortality, adhesion capacity, phagocytosis, lysosomal abundance, and oxidative burst), and transcript levels of immune-related genes involved in pathogen recognition (the Toll-like receptors, the complement system components, and the adaptor proteins MyD88) were assessed. Short-term hypoxia minimally affected hemocyte parameters, while prolonged exposure led to immunosuppression, impacting hemocyte abundance, viability, phagocytosis, and defensin gene expression. Under normoxia, nZnO stimulated immune responses of mussel hemocytes. However, combined nZnO and hypoxia induced more pronounced and rapid immunosuppression than hypoxia alone, indicating a synergistic interaction. nZnO exposure hindered immune parameter recovery during post-hypoxic reoxygenation, suggesting persistent impact. Opposing trends were observed in pathogen-sensing and pathogen-elimination mechanisms, with a positive correlation between pathogen-recognition system activation and hemocyte mortality. These findings underscore a complex relationship and potential conflict between pathogen-recognition ability, immune function, and cell survival in mussel hemocytes under hypoxia and nanopollutant stress, and emphasize the importance of considering multiple stressors in assessing the vulnerability and adaptability of mussel immune system under complex environmental conditions of anthropogenically modified coastal ecosystems.

HIF-1α and adaptor protein LIM and senescent cell antigen-like domains protein 1 axis promotes tubulointerstitial fibrosis by interacting with vimentin in angiotensin II-induced hypertension.

Activation of the renin-angiotensin system, as a hallmark of hypertension and chronic kidney diseases (CKD) is the key pathophysiological factor contributing to the progression of tubulointerstitial fibrosis. LIM and senescent cell antigen-like domains protein 1 (LIMS1) plays an essential role in controlling of cell behaviour through the formation of complexes with other proteins. Here, the function and regulation of LIMS1 in angiotensin II (Ang II)-induced hypertension and tubulointerstitial fibrosis was investigated. C57BL/6 mice were treated with Ang II to induce tubulointerstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) renal tubular-specific knockout mice or LIMS1 knockdown AAV was used to investigate their effects on Ang II-induced renal interstitial fibrosis. In vitro, HIF-1α or LIMS1 was knocked down or overexpressed in HK2 cells after exposure to Ang II. Increased expression of tubular LIMS1 was observed in human kidney with hypertensive nephropathy and in murine kidney from Ang II-induced hypertension model. Tubular-specific knockdown of LIMS1 ameliorated Ang II-induced tubulointerstitial fibrosis in mice. Furthermore, we demonstrated that LIMS1 was transcriptionally regulated by HIF-1α in tubular cells and that tubular HIF-1α knockout ameliorates LIMS1-mediated tubulointerstitial fibrosis. In addition, LIMS1 promotes Ang II-induced tubulointerstitial fibrosis by interacting with vimentin. We conclude that HIF-1α transcriptionally regulated LIMS1 plays a central role in Ang II-induced tubulointerstitial fibrosis through interacting with vimentin. Our finding represents a new insight into the mechanism of Ang II-induced tubulointerstitial fibrosis and provides a novel therapeutic target for progression of CKD.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

CED-6/GULP and Components of the Clathrin-mediated Endocytosis Machinery Act Redundantly to Correctly Display CED-1 on the Cell Membrane in C. elegans.

CED-1 is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in C. elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter includes the adapter protein CED-6/GULP and the ABC family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the AP2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

Understanding New Regulatory Mechanism of TCR Signal Transduction

Signal-transducing adaptor protein-2 (STAP-2) is a unique scaffold protein that regulates several immunological signaling pathways, including LIF/LIF receptor and LPS/TLR4 signals. STAP-2 is required for Fas/FasL-dependent T cell apoptosis and SDF-1α-induced T cell migration. Conversely, STAP-2 modulates integrin-mediated T cell adhesion, suggesting that STAP-2 is essential for several negative and positive T cell functions. However, whether STAP-2 is involved in T cell-antigen receptor (TCR)-mediated T cell activation is unknown. STAP-2 deficiency was recently reported to suppress TCR-mediated T cell activation by inhibiting LCK-mediated CD3ζ and ZAP-70 activation. Using STAP-2 deficient mice, it was demonstrated that STAP-2 is required for the pathogenesis of Propionibacterium acnes-induced granuloma formation and experimental autoimmune encephalomyelitis. Here, detailed functions of STAP-2 in TCR-mediated T cell activation, and how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases, are reviewed.

Selective targeting of mu opioid receptors to primary cilia

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both invivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.