Biology subjects

Nguyen, S. N.

Publications and source records attributed to Nguyen, S. N..

4 recordsLinked to original sources

bioRxiv2025

Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

The lncRNA Xist represents a paradigm to understand the mechanisms of RNA-mediated gene silencing in mammals, which remain largely unresolved. To induce silencing, Xist recruits the RNA-binding protein SPEN through its 5'-proximal Repeat A domain. Yet, how Repeat A recruits SPEN and how SPEN coordinates silencing remain unclear. We report that sequences in Repeat A critical for SPEN recruitment directly bind SR-rich splicing factors. SRSF1, one such factor, is required for optimal SPEN recruitment and its RS-domain recruits SPEN when tethered to Xist. SPEN and SR-protein-binding motifs promote Repeat As association with many proteins, including the m6A machinery and elongating RNA polymerase II. SPEN also represses autosomal genes where its recruitment coincides with SR-protein binding. Our results reveal an unexpectedly essential role for splicing factors in coordinating silencing by Xist and suggest that the sensing of SR-protein-rich assemblies is a general mechanism through which SPEN targets genes for repression.

genetics↗

bioRxiv2023

CD4+ T cells are homeostatic regulators during Mtb reinfection

Immunological priming - either in the context of prior infection or vaccination - elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Here, using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrate that prior Mtb infection elicits a long-lasting protective response against subsequent Mtb exposure and that the depletion of CD4+ T cells prior to Mtb rechallenge significantly abrogates this protection. Leveraging microbiologic, PET-CT, flow cytometric, and single-cell RNA-seq data from primary infection, reinfection, and reinfection-CD4+ T cell depleted granulomas, we identify differential cellular and microbial features of control. The data collectively demonstrate that the presence of CD4+ T cells in the setting of reinfection results in a reduced inflammatory lung milieu characterized by reprogrammed CD8+ T cell activity, reduced neutrophilia, and blunted type-1 immune signaling among myeloid cells, mitigating Mtb disease severity. These results open avenues for developing vaccines and therapeutics that not only target CD4+ and CD8+ T cells, but also modulate innate immune cells to limit Mtb disease.

immunology↗

bioRxiv2023

Reverse engineering of a pathogenic antibody reveals the molecular mechanism of vaccine-induced immune thrombotic thrombocytopenia

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which - vaccine-induced immune thrombotic thrombocytopenia (VITT) - is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patients blood indicates that its major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the CDR regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies, such as their ability to activate platelets in a PF4-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at Fc{gamma}RIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising polyspecificity not only sheds light on VITT etiology, but also opens up a range of opportunities to manage this pathology. Significance StatementVaccine-induced immune thrombotic thrombocytopenia (VITT) is a dangerous side effect of adenoviral-vectored vaccines that is linked to the emergence of autoantibodies recognizing platelet factor 4 (PF4). We have engineered a recombinant VITT antibody by sequencing a VITT patient-derived anti-PF4 monoclonal antibody that causes platelet activation and triggers thrombosis. This antibody was used to characterize architecture of the pathogenic immune complexes with a combination of biophysical and computational approaches, revealing the molecular mechanism of VITT. The results of this work demonstrate the critical role of electrostatics in PF4 recognition by the pathogenic antibody and the polyspecificity of the latter. Availability of the engineered VITT antibody will be invaluable for future studies aiming at understanding the general mechanistic features of autoimmune pathologies.

immunology↗

bioRxiv2023

Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT)

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but extremely dangerous side effect that has been reported for several adenoviral (Ad)-vectored COVID-19 vaccines. VITT pathology had been linked to production of antibodies that recognize platelet factor 4 (PF4), an endogenous chemokine. In this work we characterize anti-PF4 antibodies obtained from a VITT patients blood. Intact-mass MS measurements indicate that a significant fraction of this ensemble is comprised of antibodies representing a limited number of clones. MS analysis of large antibody fragments (the light chain, as well as the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire, and reveals the presence of a fully mature complex biantennary N-glycan within its Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS analysis were used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to IgG2 subclass and verify that the light chain belongs to the {lambda}-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site (absent in the germline sequence) is a result of a single mutation giving rise to an NDT motif in the antibody sequence. Peptide mapping also provides a wealth of information on lower-abundance proteolytic fragments derived from the polyclonal component of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1 through IgG4) and both types of the light chain ({lambda} and {kappa}). The structural information reported in this work will be indispensable for understanding the molecular mechanism of VITT pathogenesis.

biochemistry↗