Biology subjects

Shaffer, S. A.

Publications and source records attributed to Shaffer, S. A..

7 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

Rit2 loss in dopaminergic neurons drives a progressive Parkinsonian phenotype

Parkinsons disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.

neuroscience↗

bioRxiv2022

Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but identification and characterization of the T cell response to seasonal human coronaviruses remain largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal human coronavirus OC43. We identified MHC-I and MHC-II bound peptides derived from the viral spike, nucleocapsid, hemagglutinin-esterase, 3C-like proteinase, and envelope proteins. Only three MHC-I bound OC43-derived peptides were observed, possibly due to the potent MHC-I downregulation induced by OC43 infection. By contrast, 80 MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. These peptides elicited low-abundance recall T cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T cell lines. Among the validated epitopes, S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. N54-68 and HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow T cell cross-reactivity after infection or vaccination and could aid in the selection of epitopes for inclusion in pan-coronavirus vaccines. Author SummaryThere is much current interest in cellular immune responses to seasonal common-cold coronaviruses because of their possible role in mediating protection against SARS-CoV-2 infection or pathology. However, identification of relevant T cell epitopes and systematic studies of the T cell responses responding to these viruses are scarce. We conducted a study to identify naturally processed and presented MHC-I and MHC-II epitopes from human cells infected with the seasonal coronavirus HCoV-OC43, and to characterize the T cell responses associated with these epitopes. We found epitopes specific to the seasonal coronaviruses, as well as epitopes cross-reactive between HCoV-OC43 and SARS-CoV-2. These epitopes should be useful in following immune responses to seasonal coronaviruses and identifying their roles in COVID-19 vaccination, infection, and pathogenesis.

immunology↗

bioRxiv2022

Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, 1,3-fucosyltransferase and fucosyl 4- O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface- exposed lipid virulence factors. IMPORTANCEM. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogens ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacteria, but its existence is only known in a non- pathogenic model, M. smegmatis. Here, we demonstrated the presence of the IMD in M. tuberculosis, making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD is the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

microbiology↗

bioRxiv2021

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilize conditional Mtb knockdown mutants of essential genes as an experimentally-tractable surrogate for drug treatment, and probe the relationship between Mtb carbon metabolism and chemical-genetic interactions (CGI). We examined the anti-tubercular drugs isoniazid, rifampicin and moxifloxacin, and found that CGI are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo-relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGI could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of novel chemical-genetic-environmental interactions that can be used to optimize drug-drug interactions as well as provide a framework for understanding in vitro correlates of in vivo efficacy. SignificanceEfforts to improve tuberculosis therapy include optimizing multi-drug regimens to take advantage of drug-drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug-drug interactions. We found that these interactions can be differentially sensitive to the metabolic state and select in vitro-defined interactions can be leveraged to accelerate bacterial killing during infection. These findings suggest new strategies for optimizing tuberculosis therapy.

microbiology↗

bioRxiv2018

Resting-state functional connectivity, cortical GABA and neuroactive steroids in peripartum and peripartum depressed women: a functional magnetic imaging and resonance study

Postpartum depression (PPD) is associated with abnormalities in resting-state functional connectivity (RSFC) but the underlying neurochemistry is unclear. We hypothesized that peripartum GABAergic neuroactive steroids (NAS) are related to cortical GABA concentrations and RSFC in PPD as compared to healthy comparison women (HCW). To test this, we measured RSFC with fMRI and GABA+/Creatine (Cr) concentrations with proton magnetic resonance spectroscopy (1H MRS) in the pregenual anterior cingulate (pgACC) and occipital cortices (OCC) and quantified peripartum plasma NAS. We examined between-group differences in RSFC and the relationship between cortical GABA+/Cr concentrations with RSFC. We investigated the relationship between NAS, RSFC and cortical GABA+/Cr concentrations. Within the default mode network (DMN) an area of the dorsomedial prefrontal cortex (DMPFC) had greater connectivity with the rest of the DMN in PPD (peak voxel: MNI coordinates (2, 58, 32), p=0.002) and was correlated to depression scores (peak HAM-D17 voxel: MNI coordinates (0, 60, 34), p=0.008). pgACC GABA+/Cr correlated positively with DMPFC RSFC in a region spanning the right anterior/posterior insula and right temporal pole (r=+0.661, p=0.000). OCC GABA+/Cr correlated positively with regions spanning both amygdalae (right amygdala: r=+0.522, p=0.000; left amygdala: r=+0.651, p=0.000) as well as superior parietal areas. Plasma allopregnanolone was higher in PPD (p=0.03) and positively correlated with intra DMPFC connectivity (r=+0.548, p=0.000) but not GABA+/Cr. These results provide initial evidence that PPD is associated with altered DMN connectivity; cortical GABA+/Cr concentrations are associated with postpartum RSFC and allopregnanolone is associated with postpartum intra-DMPFC connectivity.

neuroscience↗

bioRxiv2017

C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERSTs utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology.

molecular biology↗