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Increased TLR7 signaling leading to type-I IFN production is an important contributor to human systemic lupus erythematosus (SLE). PACSIN1, a molecule that regulates synaptic vesicle recycling, has been linked to TLR7/9-mediated type I interferon production in mice, but the underlying mechanism is unknown. We undertook this study to explore pathogenicity and underlying mechanism of a de novo PACSIN1 missense variant identified in a child with SLE.PACSIN1 Q59K de novo and null variants were introduced into a human pDC cell line and mice by CRISPR/Cas9 editing. The effects of the variants on TLR7/9 signaling in human and mouse cells, as well as PACSIN1 mRNA and interferon signature in SLE patients, were assessed by real-time PCR and flow cytometry. Mechanisms were investigated by luciferase reporter assays, RNA interference, co- immunoprecipitation, and immunofluorescence.We established that PACSIN1 forms a trimolecular complex with TRAF4 and TRAF6 important for the regulation of type I-IFN. The Q59K mutation in PACSIN1 augments binding to N-WASP whilst it decreases binding to TRAF4, leading to unrestrained TRAF6-mediated activation of type-I IFN. Intriguingly, PACSIN1 Q59K increased TLR7 but not TLR9 signaling in human cells, leading to elevated IFN-β and interferon-inducible genes. Untreated SLE patients had high PACSIN1 expression in peripheral blood cells that correlated positively with interferon-related genes. Introduction of the Pacsin1 Q59K into mice caused increased surface TLR7 and Trail expression in B cells.PACSIN1 Q59K increases IFN-β activity via impairing TRAF4-mediated inhibition of TLR7 signaling, possibly contributing to SLE risk. This article is protected by copyright. All rights reserved.

Antibodies targeting aberrantly glycosylated proteins are ineffective in treating cancer. Antibody‐drug conjugates have emerged as effective alternatives, facilitating tumor‐specific drug delivery. Previous studies have assessed the aberrantly glycosylated tandem repeat region of MUC1 glycoprotein as three site‐specific glycosylated neoantigen peptide motifs (PDTR, GSTA, and GVTS) for binding with a monoclonal antibody. This study aimed to develop an antibody‐drug conjugate for cancer treatment based on monoclonal antibodies against the aforementioned three neoantigen peptide motifs. Internalization of monoclonal antibodies was assessed via immunofluorescence staining and colocalization with lysosomal markers in live cells. Antibody positivity in tumor and peritumoral tissue samples was assessed via immunohistochemistry. The efficacy of anti‐MUC1 ADCs was evaluated using various cancer cell lines and a mouse tumor xenograft model. An anti‐MUC1 ADC was synthesized by conjugating GSTA neoantigen‐specific 16A with monomethyl auristatin E (MMAE), which displayed potent antitumoral efficacy with an IC50 ranging 0.2–49.4 nM toward various cancer cells. In vivo, 16A‐MMAE inhibited tumor growth in a dose‐dependent manner in a mouse xenograft model established using the NCI‐H838 NSCLC cell line, at a minimum effective dose of 1 mg/kg. At 3 mg/kg, 16A‐MMAE did not cause significant toxicity in a transgenic mouse expressing human MUC1. The high antitumoral efficacy of 16A‐MMAE suggests that aberrant glycosylated MUC1 neoantigen is a potential target for the development of ADCs for treating various cancers. Personalized therapy may be achieved through such glycosite‐specific ADCs., In this study, we describe an anti‐MUC1 antibody‐drug conjugate, 16A‐monomethyl auristatin E (MMAE). 16A‐MMAE showed potent antitumoral activity in multiple types of cancer, suggesting that neoantigens generated by posttranslational modification are promising targets for antibody‐drug development.

Somatic mutations in P2RY8 that promote B cell growth and migration are common in lymphomas. He et al. describe germline loss-of-function P2RY8 variants in SLE and uncover novel functions of P2RY8 in immunological tolerance through restraining plasma cell development and promoting B cell–negative selection., B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a “de novo” variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis., Graphical Abstract

Background Resistance to thyroid hormones is a very rare condition, which is often misdiagnosed and mistreated. The cases where there is a concomitant autoimmune thyroid disorder are ultra-rare and particularly challenging to treat. Diagnostic and research-based genomic testing can sometimes identify pathogenic variants unrelated to the primary reason for testing (incidental findings). Case presentation We present a patient with thyroid resistance associated with hypothyroid Hashimoto thyroiditis. The long diagnostic odyssey spanning over 20-years included repeated misdiagnoses and mistreatments and was concluded by a research-based genomic testing, identifying a “de novo” THRB pathogenic variant. The varying sensitivity of various tissues to thyroid hormones accompanied by hypothyroid Hashimoto thyroiditis continues to pose a significant treatment challenge. Conclusions Thyroid hormone resistance continues to be an un(der)- and misdiagnosed thyroid condition whose management is particularly challenging when associated with autoimmune thyroid disease. Whole exome sequencing has the potential to identify THRB pathogenic variants as incidental findings. Reporting such secondary findings from genomic testing may be particularly important in the context of the rarity of the condition and the potential clinical consequences of misdiagnosis and mistreatment.

BackgroundAntibodies targeting abnormally glycosylated proteins have been ineffective in treating cancer. Antibody-drug conjugates are emerging as an efficient option, which allow specific delivery of drugs into tumors. We and others have dissected the abnormally glycosylated tandem repeat region of MUC1 glycoprotein as three site-specific glycosylated neoantigen peptide motifs (PDTR, GSTA, GVTS) for monoclonal antibody binding.MethodsInternalization of monoclonal antibodies was studied by immunofluorescence staining and colocalization with lysosomal markers in live cells. Antibody positivity in tumor and peritumoral tissue samples were studied by immunohistochemistry. The efficacy of anti-MUC1 ADCs were evaluated with various cancer cell lines and mouse tumor xenograft model.ResultsWe describe an anti-MUC1 ADC by conjugating GSTA neoantigen-specific 16A with monomethyl auristatin E (MMAE). 16A-MMAE showed potent antitumoral efficacy with IC50 ranging from 0.2 to 49.4 nM toward multiple types of cancer cells. In vivo, 16A-MMAE showed dose-dependent inhibition of tumor growth in mouse xenograft of NCI-H838 NSCLC cell line, with minimum effective dose at 1 mg/kg. At the dose of 3 mg/kg, 16A-MMAE did not cause significant toxicity in a transgenic mouse expressing human MUC1.ConclusionsThe high antitumoral efficacy of 16A-MMAE suggest that aberrant glycosylated MUC1 neoantigen is a target with high positivity in multiple cancer types for ADC development. Personalized therapy may be achieved by development of glycosite-specific antibody-drug conjugates.

Environmental stresses are important factors causing male infertility which attracts broad attention. Protein acetylation is a pivotal post-translational modification and modulates diverse physiological processes including spermatogenesis. In this study, we employed quantitative proteomic techniques and bioinformatics tools to analyze the alterations of acetylome profile of mouse testis after heat shock and X-irradiation. Overall, we identified 1139 lysine acetylation sites in 587 proteins in which 1020 lysine acetylation sites were quantified. The Gene Ontology analysis showed that the major acetylated protein groups were involved in generation of precursor metabolites and metabolic processes, and were localized predominantly in cytosolic and mitochondrial. Compared to the control group, 36 sites of 28 acetylated proteins have changed after heat shock, and 49 sites of 43 acetylated proteins for X-ray exposure. Some of the differentially acetylated proteins have been reported to be associated with the progression of spermatogenesis and male fertility. We observed the up-regulated acetylation level change on testis specific histone 2B and heat shock protein upon heat treatment and a sharp decline of acetylation level on histone H2AX under X-ray treatment, suggesting their roles in male germ cells. Notably, the acetylation level on K279 of histone acetyltransferase (Kat7) was down-regulated in both heat and X-ray treatments, indicating that K279 may be a key acetylated site and affect its functions in spermatogenesis. Our results reveal that protein acetylation might add another layer of complexity to the regulation for spermatogenesis, and further functional studies of these proteins will help us elucidate the mechanisms of abnormal spermatogenesis.

Human Huntingtin (HTT), a Huntington's disease gene, is highly expressed in the mammalian brain and testis. Simultaneous knockout of mouse Huntingtin (Htt) in brain and testis impairs male fertility, providing evidence for a link between Htt and spermatogenesis; however, the underlying mechanism remains unclear. To understand better the function of Htt in spermatogenesis, we restricted the genetic deletion specifically to the germ cells using the Cre/loxP site-specific recombination strategy and found that the resulting mice manifested smaller testes, azoospermia and complete male infertility. Meiotic chromosome spread experiments showed that the process of meiosis was normal in the absence of Htt. Notably, we found that Htt-deficient round spermatids did not progress beyond step 3 during the post-meiotic phase, when round spermatids differentiate into mature spermatozoa. Using an iTRAQ-based quantitative proteomic assay, we found that knockout of Htt significantly altered the testis protein profile. The differentially expressed proteins exhibited a remarkable enrichment for proteins involved in translation regulation and DNA packaging, suggesting that Htt might play a role in spermatogenesis by regulating translation and DNA packaging in the testis.