The rockefeller university

Motivated by recent data pointing to the existence of homo- oligomeric assemblies of membrane proteins called higher- order transient structures, and their apparent role in connecting components of membrane signal pathways, we examine here by cryoelectron microscopy some of the protein-protein interactions that occur in cluster formation. Metabotropic glutamate receptors and HCN ion channels inside clusters contact their neighbors through structured extracellular and intracellular domains, respectively. Other ion channels, including Kv2.1 and Slo1, appear to form clusters through prominent intrinsically disordered sequences in the cytoplasm. These distinct modes of interaction are associated with clusters exhibiting varying degrees of compactness and order. We conclude that nature utilizes a variety of ways to form connections between membrane proteins in self- assembled clusters.

Postmitotic neurons have high levels of methylated cytosine and its oxidized intermediates such as 5-hydroxymethylcytosine1. However, the functional relevance of these epigenetic modifications of DNA are poorly understood. Here we show that some cytidine analogues, such as cytarabine, cause DNA double-strand breaks during TET-mediated active 5-methylcytosine demethylation by interrupting TDG-dependent base excision repair. These double-strand breaks are frequently converted into deletions and translocations by DNA ligase 4. In vivo, Purkinje and Golgi cells in the cerebellum are the only neuronal populations that exhibit high levels of DNA damage due to cytarabine. In Purkinje cells, TET targets highly expressed gene bodies marked by enhancer-associated histone modifications. Many of these genes control movement coordination, which explains the long-recognized cerebellar neurotoxicity of cytarabine2. We show that other cytidine analogues, such as gemcitabine, cause only single-strand breaks in neurons, which are repaired by DNA ligase 3 with minimal toxicity. Our findings uncover a mechanistic link between TET-mediated DNA demethylation, base excision repair and gene expression in neurons. The results also provide a rational explanation for the different neurotoxicity profiles of an important class of antineoplastic agents.

Aim: Although several SERPINA1 genetic variants have been reported for their pathogenicity to induce liver disorders through phenotype-driven approaches, data regarding genotype-driven approaches of the SERPINA1 locus remain unavailable. This study aimed to characterize the clinical and liver biological profiles of patients harboring nonbenign SERPINA1 variants. Methods: We conducted a retrospective, exome-based genotype-first reverse phenotyping study using structured electronic health record data from consecutive patients from January 1, 2015, to January 31, 2022. Statistical associations were assessed using frequentist and Bayesian models, with validation in the UK Biobank cohort. Results: Among 1377 patients analyzed, 15 SERPINA1 variants classified as nonbenign were identified in 217 (15.7%) patients. Data were available for 126 patients (median age, 41.5 years; 52.4% male). Liver disease, hyperferritinemia, and pulmonary emphysema were observed in 32.5% (41/126), 23% (29/126), and 5.6% (7/126) of the patients, respectively. The median follow-up duration was 1.3 years and encompassed 1085 biological observations. We confirmed associations with well-documented variants of SERPINA1 (p.Glu366Lys, p.Pro393Ser, p.Ala308Ser, p.Glu288Val, and p.Phe76del). We identified three novel genetic associations with liver markers: c.*10G > A, c.1065 + 10C > T, and p.Arg63Cys. The UK Biobank data confirmed significant gene- and variant-level associations, notably for the variants identified in our study, which ranked in the top decile of statistical associations. Conclusions: This study supports the utility of a genotype-first approach in characterizing hepatic manifestations of nonbenign SERPINA1 variants. The findings highlight novel genotype-biomarker associations and suggest a role for SERPINA1 genetic testing in patients with unexplained liver abnormalities.

A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.

Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome-wide essentiality profiles in bacteria and has been extensively applied to Mycobacterium tuberculosis (Mtb). Dozens of TnSeq screens have yielded valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. Despite their value, these Mtb TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in Mtb. We address this problem by building a central repository of publicly available Mtb TnSeq screens, the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date approximate to 150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict the functional annotation of orphan genes in Mtb. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of Mtb genes, and help predict gene function in this important human pathogen.

Colibactin, a human microbiome-derived genotoxin, promotes colorectal cancer by damaging the host gut epithelial genomes. While colibactin is synthesized via a hybrid non-ribosomal peptide synthetase (NRPS)polyketide synthase (PKS) pathway, known as pks or clb, the structural details of its biosynthetic enzymes remain limited, hindering our understanding of its biosynthesis and clinical application. In this study, we report the cryo-EM structures of two colibactin-producing PKS enzymes, ClbC and ClbI, captured in different reaction states using a substrate-mimic crosslinker. Our structural analysis revealed the binding sites of carrier protein (CP) domains of the ClbC and ClbI on their ketosynthase (KS) domains. Further, we identified a novel NRPS-PKS docking interaction between ClbI and its upstream enzyme, ClbH, mediated by the C-terminal peptide ClbH and the dimeric interface of ClbI, establishing a 1:2 stoichiometry. These findings advance our understanding of colibactin assembly line and provide broader insights into NRPS-PKS natural product biosynthesis mechanisms.

Lung cancer continues to be the leading cause of cancer-related deaths globally. Unraveling the regulators behind lung cancer growth and its metastatic spread, along with understanding the underlying mechanisms, is crucial for developing novel and effective therapeutic strategies. While much research has focused on identifying potential oncogenes or tumor suppressors, the roles of certain genes can vary depending on the context and may even exhibit contradictory effects. In this study, we demonstrate that acyl-CoA binding domain containing 3 (ACBD3), a Golgi resident protein, promotes primary lung cancer growth by recruiting phosphatidylinositol (PI)-4-kinase III beta (PI4KB) to the Golgi, thereby enhancing oncogenic secretion in chromosome 1q-amplified lung cancer cells. Conversely, in chromosome 1q-diploid lung cancer cells, ACBD3 acts as a suppressor of lung cancer metastasis by inhibiting the NOTCH signaling pathway and reducing cancer cell motility. This highlights the intricacy of cancer progression and cautions against simplistic approaches targeting individual oncogenes for cancer therapy.

Endothelial cells form the inner layer of blood vessels and play key roles in circulatory system development and function. A variety of endothelial cell types have been described through gene expression and transcriptome studies; nonetheless, the transcriptional programs that specify endothelial cell fate and maintenance are not well understood. To uncover such regulatory programs, we studied the C. elegans head mesodermal cell (HMC), a noncontractile mesodermal cell bearing molecular and functional similarities to vertebrate endothelial cells. Here, we demonstrate that a Forkhead transcription factor, LET-381, is required for HMC fate specification and maintenance of HMC gene expression. DMD-4, a DMRT transcription factor, acts downstream of and in conjunction with LET-381 to mediate these functions. Independently of LET-381, DMD-4 also represses the expression of genes associated with a different, non-HMC, mesodermal fate. Our studies uncover essential roles for FoxF transcriptional regulators in endothelial cell development and suggest that FoxF co-functioning target transcription factors promote specific non-contractile mesodermal fates.

Nguni sheep (Ovis aries) are indigenous to the Southern Africa region and common within the smallholder and poor resources farming systems. They are well adapted to different agroecological regions. However, limited genomic resources such as high-quality reference genomes have hindered our understanding of its adaptation and establishment of an effective breeding program. To address this, we assembled a chromosomal-level genome of Nguni sheep using a combination of PacBio HiFi reads and Omni-C reads. The genome size was estimated to be 2.9 Gb with a contig/scaffold N50 74 Mb and 99.6 Mb and a genome completeness of 96.1%, as estimated by the Benchmarking Universal Single-Copy Orthologs (BUSCO) program. The final genome encompassed a total of 25,926 protein-coding genes. The findings of this study provide a valuable genomic resource for understanding the adaptability of the Nguni sheep and the establishment of effective breeding programs.