The rockefeller university

Protein S-palmitoylation, or S-fatty-acylation, regulates many fundamental cellular processes in eukaryotes. Herein, we present a chemical fatty-acylation approach that involves site-specific incorporation of cycloalkyne-containing unnatural amino acids and subsequent bioorthogonal reactions with fatty-acyl tetrazines to install fatty-acylation mimics at target protein sites, allowing gain-of-function analysis of S-palmitoylation in live cells.

We each developed from a single cell—a fertilized egg—that divided and divided and eventually gave rise to the trillions of cells, of hundreds of types, that constitute the tissues and organs of our adult bodies. Advancing our understanding of the molecular programs underlying the emergence and differentiation of these diverse cell types is of fundamental interest and will affect almost every aspect of biology and medicine.

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G(2)/M cell cycle arrest. Vif initiates G(2)/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-beta/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis. IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G(2)/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G(2)/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling similar to 4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

Polyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores, and others. Despite their involvement in bacterium-bacterium and bacterium-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics, and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low-similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic data sets revealed higher abundances of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs. IMPORTANCE We identified distinct secondary-metabolite-encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary-metabolite-encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in interbacterial and bacterium-plant interactions.

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.

Keloids are disfiguring, fibroproliferative growths and their pathogenesis remains unclear, inhibiting therapeutic development. Available treatment options have limited efficacy and harbor safety concerns. Thus, there is a great need to clarify keloid pathomechanisms that may lead to novel treatments. In this study, we aimed to elucidate the profile of lesional and non-lesional keloid skin compared to normal skin. We performed gene (RNAseq, qRT-PCR) and protein (immunohistochemistry) expression analyses on biopsy specimens obtained from lesional and non-lesional skin of African American (AA) keloid patients compared to healthy skin from AA controls. Fold-change >= 2 and false-discovery rate (FDR)<0.05 was used to define significance. We found that lesional versus normal skin showed significant up-regulation of markers of T-cell activation/migration (ICOS, CCR7), Th2- (IL-4R, CCL11, TNFSF4/OX40L), Th1- (CXCL9/CXCL10/CXCL11), Th17/Th22- (CCL20, S100As) pathways, and JAK/STAT-signaling (JAK3) (false-discovery rate [FDR]<0.05). Non-lesional skin also exhibited similar trends. We observed increased cellular infiltrates in keloid tissues, including T-cells, dendritic cells, mast cells, as well as greater IL-4r alpha(+), CCR9(+), and periostin(+) immunostaining. In sum, comprehensive molecular profiling demonstrated that both lesional and non-lesional skin show significant immune alternations, and particularly Th2 and JAK3 expression. This advocates for the investigation of novel treatments targeting the Th2 axis and/or JAK/STAT-signaling in keloid patients.

Heterozygous gain-of-function (GOF) mutations inSTAT1in patients with chronic mucocutaneous candidiasis (CMC) and hypothyroidism were discovered in 2011. CMC is the recurrent or persistent mucocutaneous infection byCandidafungi, and hypothyroidism results from autoimmune thyroiditis. Patients with these diseases develop other infectious diseases, including viral, bacterial, and fungal diseases, and other autoimmune manifestations, including enterocolitis, immune cytopenia, endocrinopathies, and systemic lupus erythematosus.STAT1-GOF mutations are highly penetrant with a median age at onset of 1 year and often underlie an autosomal dominant trait. As many as 105 mutations at 72 residues, including 65 recurrent mutations, have already been reported in more than 400 patients worldwide. The GOF mechanism involves impaired dephosphorylation of STAT1 in the nucleus. Patient cells show enhanced STAT1-dependent responses to type I and II interferons (IFNs) and IL-27. This impairs Th17 cell development, which accounts for CMC. The pathogenesis of autoimmunity likely involves enhanced type I IFN responses, as in other type I interferonopathies. The pathogenesis of other infections, especially those caused by intramacrophagic bacteria and fungi, which are otherwise seen in patients with diminished type II IFN immunity, has remained mysterious. The cumulative survival rates of patients with and without severe disease (invasive infection, cancer, and/or symptomatic aneurysm) at 60 years of age are 31% and 87%, respectively. Severe autoimmunity also worsens the prognosis. The treatment of patients withSTAT1-GOF mutations who suffer from severe infectious and autoimmune manifestations relies on hematopoietic stem cell transplantation and/or oral JAK inhibitors.

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4 (+)CD8 alpha alpha (+) IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4 (+) T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4 (+)CD8 alpha alpha (+) IEL differentiation. However, TCR ablation on differentiated CD4 (+)CD8 alpha alpha (+) IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4 (+)CD8 alpha alpha (+) IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4 (+) T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.

Nutrient limitation drives competition for resources across organisms. However, much is unknown about how selective pressures resulting from nutrient limitation shape microbial coding sequences. Here, we study this "resource-driven selection" by using metagenomic and single-cell data of marine microbes, alongside environmental measurements. We show that a significant portion of the selection exerted on microbes is explained by the environment and is associated with nitrogen availability. Notably, this resource conservation optimization is encoded in the structure of the standard genetic code, providing robustness against mutations that increase carbon and nitrogen incorporation into protein sequences. This robustness generalizes to codon choices from multiple taxa across all domains of life, including the human genome.