Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease.

The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.

Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism.

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.

Translational Control by 4E-BP1/2 Suppressor Proteins Regulates Mitochondrial Biosynthesis and Function during CD8+ T Cell Proliferation.

CD8+ T cell proliferation and differentiation into effector and memory states are high-energy processes associated with changes in cellular metabolism. CD28-mediated costimulation of T cells activates the PI3K/AKT/mammalian target of rapamycin signaling pathway and induces eukaryotic translation initiation factor 4E-dependent translation through the derepression by 4E-BP1 and 4E-BP2. In this study, we demonstrate that 4E-BP1/2 proteins are required for optimum proliferation of mouse CD8+ T cells and the development of an antiviral effector function. We show that translation of genes encoding mitochondrial biogenesis is impaired in T cells derived from 4E-BP1/2-deficient mice. Our findings demonstrate an unanticipated role for 4E-BPs in regulating a metabolic program that is required for cell growth and biosynthesis during the early stages of CD8+ T cell expansion.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

Pharmacologic inhibition of PARP5, but not that of PARP1 or 2, promotes cytokine production and osteoclastogenesis through different pathways.

OBJECTIVES: PARPs, which are members of the poly(ADP-ribose) polymerase superfamily, promote tumorigenesis and tumour-associated inflammation and are thus therapeutic targets for several cancers. The aim of the present study is to investigate the mechanistic insight into the roles PARPs for inflammation. METHODS: Primary murine macrophages were cultured in the presence or absence of the PARP5 inhibitor NVP-TNKS656 to examine the role of PARP5 for cytokine production. RESULTS: In contrast to the roles of other PARPs for induction of inflammation, we found in the present study that pharmacologic inhibition of PARP5 induces production of inflammatory cytokines in primary murine macrophages. We found that treatment with the PARP5 inhibitor NVP-TNKS656 in macrophages enhanced steady-state and LPS-mediated cytokine production through degradation of IκBα and subsequent nuclear translocation of NF-κB. We also found that pharmacologic inhibition of PARP5 stabilises the adaptor protein 3BP2, a substrate of PARP5, and that accelerated cytokine production induced by PARP5 inhibition was rescued in 3BP2-deleted macrophages. Additionally, we found that LPS increases the expression of 3BP2 and AXIN1, a negative regulator of ß-catenin, through suppression of PARP5 transcripts in macrophages, leading to further activation of cytokine production and inhibition of ß-catenin-mediated cell proliferation, respectively. Lastly, we found that PARP5 inhibition in macrophages promotes osteoclastogenesis through stabilisation of 3BP2 and AXIN1, leading to activation of SRC and suppression of ß-catenin, respectively. CONCLUSIONS: Our results show that pharmacologic inhibition of PARP5 against cancers unexpectedly induces adverse autoinflammatory side effects through activation of innate immunity, unlike inhibition of other PARPs.

GEF-H1 Is Required for Colchicine Inhibition of Neutrophil Rolling and Recruitment in Mouse Models of Gout.

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1-/-) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1-/- mice. Inhibition of macrophage IL-1ß production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions.

The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2.

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.

Preexisting autoimmune disease and immune-related adverse events associated with anti-PD-1 cancer immunotherapy: a national case series from the Canadian Research Group of Rheumatology in Immuno-Oncology.

BACKGROUND: Limited data are available on the safety and efficacy of immune checkpoint inhibitors (ICI) in patients with preexisting autoimmune diseases (PAD). METHODS: Retrospective study of patients with PAD referred for rheumatologic evaluation prior to starting or during immunotherapy between January 2013 and July 2019 from 10 academic sites across Canada. Data were extracted by chart review using a standardized form. RESULTS: Twenty-seven patients with PAD on ICI therapy were identified. The most common PADs were rheumatoid arthritis (30%), psoriasis/psoriatic arthritis (30%), inflammatory bowel disease (IBD, 15%) and axial spondyloarthritis (11%), and the most frequently observed cancers were lung cancer and melanoma. All patients received anti-PD-1 therapies, and 2 received additional sequential anti-CTLA-4 therapy. PAD exacerbations occurred in 52% over a median (IQR) follow-up of 11.0 (6.0-17.5) months, with 14% being severe, 57% requiring corticosteroids, 50% requiring immunosuppression and 14% requiring ICI discontinuation. Flares were generally more frequent and severe in patients who previously required more intensive immunosuppression (i.e., biologics). Flares occurred despite background immunosuppression at the time of ICI initiation. In patients with preexisting psoriasis, IBD and axial spondyloarthritis, rheumatic immune-related adverse events (irAEs), mostly polyarthritis and tenosynovitis, were frequently observed. Tumor progression was not associated with exposure to immunosuppressive drugs before or after ICI initiation and was numerically less frequent in patients with irAEs. CONCLUSION: PAD exacerbations in the context of ICI treatment are common, although generally mild, and occur despite background immunosuppression. Exacerbations are more frequent and severe in patients on more intensive immunosuppressive therapies pre-immunotherapy.

Anticardiolipin and other antiphospholipid antibodies in critically ill COVID-19 positive and negative patients.

BACKGROUND: Reports of severe COVID-19 being associated with thrombosis, antiphospholipid antibodies (APLA), and antiphospholipid syndrome have yielded disparate conclusions. Studies comparing patients with COVID-19 with contemporaneous controls of similar severity are lacking. METHODS: 22 COVID-19+ and 20 COVID-19- patients with respiratory failure admitted to intensive care were studied longitudinally. Demographic and clinical data were obtained from the day of admission. APLA testing included anticardiolipin (aCL), anti-ß2glycoprotien 1 (ß2GP1), antidomain 1 ß2GP1 and antiphosphatidyl serine/prothrombin complex. Antinuclear antibodies (ANAs) were detected by immunofluorescence and antibodies to cytokines by a commercially available multiplexed array. Analysis of variance was used for continuous variables and Fisher's exact test was used for categorical variables with α=0.05 and the false discovery rate at q=0.05. RESULTS: APLAs were predominantly IgG aCL (48%), followed by IgM (21%) in all patients, with a tendency towards higher frequency among the COVID-19+. aCL was not associated with surrogate markers of thrombosis but IgG aCL was strongly associated with worse disease severity and higher ANA titres regardless of COVID-19 status. An association between aCL and anticytokine autoantibodies tended to be higher among the COVID-19+. CONCLUSIONS: Positive APLA serology was associated with more severe disease regardless of COVID-19 status. TRIAL REGISTRATION NUMBER: NCT04747782.

Low junctional adhesion molecule-A expression is associated with an epithelial to mesenchymal transition and poorer outcomes in high-grade serous carcinoma of uterine adnexa.

High-grade serous carcinoma of uterine adnexa (HGSC) is the most frequent histotype of epithelial ovarian cancer and has a poor 5-year survival rate due to late-stage diagnosis and the poor efficacy of standard treatments. Novel biomarkers of cancer outcome are needed to identify new targetable pathways and improve personalized treatments. Cell-surface screening of 26 HGSC cell lines by high-throughput flow cytometry identified junctional adhesion molecule 1 (JAM-A, also known as F11R) as a potential biomarker. Using a multi-labeled immunofluorescent staining coupled with digital image analysis, protein levels of JAM-A were quantified in tissue microarrays from three HGSC patient cohorts: a discovery cohort (n = 101), the Canadian Ovarian Experimental Unified Resource cohort (COEUR, n = 1158), and the Canadian Cancer Trials Group OV16 cohort (n = 267). Low JAM-A level was associated with poorer outcome in the three cohorts by Kaplan-Meier (p = 0.023, p < 0.001, and p = 0.036, respectively) and was an independent marker of shorter survival in the COEUR cohort (HR = 0.517 (0.381-703), p < 0.001). When analyses were restricted to patients treated by taxane-platinum-based chemotherapy, low JAM-A protein expression was associated with poorer responses in the COEUR (p < 0.001) and OV16 cohorts (p = 0.006) by Kaplan-Meier. Decreased JAM-A gene expression was an indicator of poor outcome in gene expression datasets including The Cancer Genome Atlas (n = 606, p = 0.002) and Kaplan-Meier plotter (n = 1816, p = 0.024). Finally, we observed that tumors with decreased JAM-A expression exhibited an enhanced epithelial to mesenchymal transition (EMT) signature. Our results demonstrate that JAM-A expression is a robust prognostic biomarker of HGSC and may be used to discriminate tumors responsive to therapies targeting EMT.

The LUBAC participates in lysophosphatidic acid-induced NF-κB activation.

The natural bioactive glycerophospholipid lysophosphatidic acid (LPA) binds to its cognate G protein-coupled receptors (GPCRs) on the cell surface to promote the activation of several transcription factors, including NF-κB. LPA-mediated activation of NF-κB relies on the formation of a signalosome that contains the scaffold CARMA3, the adaptor BCL10 and the paracaspase MALT1 (CBM complex). The CBM complex has been extensively studied in lymphocytes, where it links antigen receptors to NF-κB activation via the recruitment of the linear ubiquitin assembly complex (LUBAC), a tripartite complex of HOIP, HOIL1 and SHARPIN. Moreover, MALT1 cleaves the LUBAC subunit HOIL1 to further enhance NF-κB activation. However, the contribution of the LUBAC downstream of GPCRs has not been investigated. By using murine embryonic fibroblasts from mice deficient for HOIP, HOIL1 and SHARPIN, we report that the LUBAC is crucial for the activation of NF-κB in response to LPA. Further echoing the situation in lymphocytes, LPA unbridles the protease activity of MALT1, which cleaves HOIL1 at the Arginine 165. The expression of a MALT1-insensitive version of HOIL1 reveals that this processing is involved in the optimal production of the NF-κB target cytokine interleukin-6. Lastly, we provide evidence that the guanine exchange factor GEF-H1 favors MALT1-mediated cleavage of HOIL1 and NF-κB signaling in this context. Together, our results unveil a critical role for the LUBAC as a positive regulator of NF-κB signaling downstream of LPA receptors.

Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition.

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

Multiplexed Real-Time NMR GTPase Assay for Simultaneous Monitoring of Multiple Guanine Nucleotide Exchange Factor Activities from Human Cancer Cells and Organoids.

Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.

Measuring error rates in genomic perturbation screens: gold standards for human functional genomics.

Technological advancement has opened the door to systematic genetics in mammalian cells. Genome-scale loss-of-function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off-target effects of perturbation reagents and poorly quantified error rates in large-scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold-standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.

Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2.

The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated ß-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities.

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

The 3BP2 adapter protein is required for chemoattractant-mediated neutrophil activation.

3BP2 is a pleckstrin homology and Src homology 2 domain-containing adapter protein mutated in cherubism, a rare autosomal-dominant human bone disorder. Previously, we have demonstrated a functional role for 3BP2 in peripheral B cell development and in peritoneal B1 and splenic marginal zone B cell-mediated Ab responses. In this study, we show that 3BP2 is required for G protein-coupled receptor-mediated neutrophil functions. Neutrophils derived from 3BP2-deficient (Sh3bp2-/-) mice failed to polarize their actin cytoskeleton or migrate in response to a gradient of chemotactic peptide, fMLF. Sh3bp2-/- neutrophils failed to adhere, crawl, and emigrate out of the vasculature in response to fMLF superfusion. 3BP2 is required for optimal activation of Src family kinases, small GTPase Rac2, neutrophil superoxide anion production, and for Listeria monocytogenes bacterial clearance in vivo. The functional defects observed in Sh3bp2-/- neutrophils may partially be explained by the failure to fully activate Vav1 guanine nucleotide exchange factor and properly localize P-Rex1 guanine nucleotide exchange factor at the leading edge of migrating cells. Our results reveal an obligate requirement for the adapter protein 3BP2 in G protein-coupled receptor-mediated neutrophil function.

Scaffold protein SH3BP2 signalosome is pivotal for immune activation in nephrotic syndrome.

Despite clinical use of immunosuppressive agents, the immunopathogenesis of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remains unclear. Src homology 3-binding protein 2 (SH3BP2), a scaffold protein, forms an immune signaling complex (signalosome) with 17 other proteins, including phospholipase Cγ2 (PLCγ2) and Rho-guanine nucleotide exchange factor VAV2 (VAV2). Bioinformatic analysis of human glomerular transcriptome (Nephrotic Syndrome Study Network cohort) revealed upregulated SH3BP2 in MCD and FSGS. The SH3BP2 signalosome score and downstream MyD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS. Immune pathway activation scores for Toll-like receptors, cytokine-cytokine receptor, and NOD-like receptors were increased in FSGS. Lower SH3BP2 signalosome score was associated with MCD, higher estimated glomerular filtration rate, and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6-fold and ~25-fold increases in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron-dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes, underscoring the importance of SH3BP2 in immune activation. SH3BP2 and its binding partners may determine the immune activation pathways resulting in podocyte injury leading to loss of the glomerular filtration barrier.

Modulation of IL-7 thresholds by SOCS proteins in developing B lineage cells.

During B lymphopoiesis, IL-7 induces survival, proliferation, and differentiation signals that are important during the pro-B to pre-B cell transition. We showed that murine small pre-B stage cells do not signal or proliferate in response to IL-7, yet they maintain IL-7R surface expression. Loss of proliferative responsiveness to IL-7 is mediated by suppressor of cytokine signaling protein 1 (SOCS-1), the expression of which is regulated during B lymphopoiesis, with the highest levels observed in small pre-B cells. SOCS-1 inhibits IL-7 responses in pre-B cell lines and ex vivo B lineage cells. SOCS-1 expression and, thus, responsiveness to IL-7, can be regulated by IL-7 itself, as well as IFN-γ and IL-21. Additionally, the transcriptional repressor Gfi-1b enhances the proliferative responsiveness of B cell lines to IL-7. We demonstrated that these molecules act together to form a SOCS-mediated "rheostat" that controls the level of IL-7R signaling in developing murine B lineage cells.