The rockefeller university

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of similar to 150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type's specification, as well as 'cell-type homologs' across vertebrate evolution.

Aberrant cleavage of amyloid precursor protein (APP) by gamma-secretase is closely associated with Alzheimer's disease (AD). gamma-secretase activating protein (GSAP) specifically promotes gamma-secretase-mediated cleavage of APP. However, the underlying mechanism remains enigmatic. Here, we demonstrate that the 16-kDa C-terminal fragment of GSAP (GSAP-16K) undergoes phase separation in vitro and forms puncta-like condensates in cells. GSAP-16K exerts dual modulation on gamma-secretase cleavage; GSAP-16K in dilute phase increases APP-C-terminal 99-residue fragment (C99) cleavage toward preferred production of p-amyloid peptide 42 (A beta 42), but GSAP-16K condensates reduce APP-C99 cleavage through substrate sequestration. Notably, the A beta 42/A beta 40 ratio is markedly elevated with increasing concentrations of GSAP-16K. GSAP-16K stably associates with APP-C99 through specific sequence elements. These findings mechanistically explain GSAP-mediated modulation of gamma-secretase activity that may have ramifications on the development of potential therapeutics.

Prokaryotic organisms have developed multiple defense systems against phages; however, little is known about whether and how these interact with each other. Here, we studied the connection between two of the most prominent prokaryotic immune systems: restriction-modification and CRISPR. While both systems employ enzymes that cleave a specific DNA sequence of the invader, CRISPR nucleases are programmed with phage-derived spacer sequences, which are integrated into the CRISPR locus upon infection. We found that restriction endonucleases provide a short-term defense, which is rapidly overcome through methylation of the phage genome. In a small fraction of the cells, however, restriction results in the acquisition of spacer sequences from the cleavage site, which mediates a robust type II-A CRISPR-Cas immune response against the methylated phage. This mechanism is reminiscent of eukaryotic immunity in which the innate response offers a first temporary line of defense and also activates a second and more robust adaptive response.

Tumourigenesis and cancer progression require enhanced global protein translation(1-3). Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery(4,5). Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase-along with its downstream cognate tRNAs-elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease that presents as exquisitely tender abscesses, draining fistulae, and sinus tracts. HS can lead to significant impairments in patients' quality of life, especially for children and adolescents who face challenges related to self-esteem and physical and emotional development. Severe long-term physical sequelae of inadequately treated HS include extensive scarring, urogenital strictures, immobility, and squamous cell carcinoma; emotional sequelae include depression, anxiety, and suicidal ideation. Many of the devastating long-term sequelae associated with HS can be prevented with early recognition and proper collaborative management. This article reviews strategies to aid pediatricians in early diagnosis of HS and provides clinical pearls for management and prevention of disease flares.

Inward rectifier K+(Kir) channels regulate cell membrane potential. Different Kir channels respond to unique ligands, but all are regulated by phosphatidylinositol 4,5bisphosphate (PI(4,5)P-2). Using planar lipid bilayers, we show that Kir2.2 exhibits bursts of openings separated by long quiescent interburst periods. Increasing PI(4,5)P-2 concentration shortens the Kir2.2 interburst duration and lengthens the burst duration without affecting dwell times within a burst. From this, we propose that burst and interburst durations correspond to the cytoplasmic domain (CTD)-docked and CTDundocked conformations observed in the presence and absence of PI(4,5)P-2 in atomic structures. We also studied the effect of different phosphatidylinositol lipids on Kir2.2 activation and conclude that the 5' phosphate is essential to Kir2.2 pore opening. Other phosphatidylinositol lipids can compete with PI(4,5)P-2 but cannot activate Kir2.2 without the 5' phosphate. PI(4)P, which is directly interconvertible to and from PI(4,5)P-2, might thus be a regulator of Kir channels in the plasma membrane.

Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.

Alpha-2-Macroglobulin (A2M) is the critical pan-protease inhibitor of the innate immune system. When proteases cleave the A2M bait region, global structural transformation of the A2M tetramer is triggered to entrap the protease. The structural basis behind the cleavage-induced transformation and the protease entrapment remains unclear. Here, we report cryo-EM structures of native-and intermediate-forms of the Xenopus laevis egg A2M homolog (A2Moo or ovomacroglobulin) tetramer at 3.7-4.1 A & DBLBOND; and 6.4 A & DBLBOND; resolution, respectively. In the native A2Moo tetramer, two pairs of dimers arrange into a cross-like configuration with four 60 & DBLBOND;A-wide bait-exposing grooves. Each bait in the native form threads into an aperture formed by three macroglobulin domains (MG2, MG3, MG6). The bait is released from the narrowed aperture in the induced protomer of the intermediate form. We propose that the intact bait region works as a "latch-lock " to block futile A2M transformation until its protease-mediated cleavage. (C) 2021 Elsevier Ltd. All rights reserved.

Human USP18 is an interferon (IFN)-stimulated gene product and a negative regulator of type I IFN (IFN-I) signaling. It also removes covalently linked ISG15 from proteins, in a process called deISGylation. In turn, ISG15 prevents USP18 from being degraded by the proteasome. Autosomal recessive complete USP18 deficiency is life-threatening in infancy owing to uncontrolled IFN-I-mediated autoinflammation. We report three Moroccan siblings with autoinflammation and mycobacterial disease who are homozygous for a new USP18 variant. We demonstrate that the mutant USP18 (p.I60N) is normally stabilized by ISG15 and efficient for deISGylation but interacts poorly with the receptor-anchoring STAT2 and is impaired in negative regulation of IFN-I signaling. We also show that IFN-gamma-dependent induction of IL-12 and IL-23 is reduced owing to IFN-I-mediated impairment of myeloid cells to produce both cytokines. Thus, insufficient negative regulation of IFN-I signaling by USP18-I60N underlies a specific type I interferonopathy, which impairs IL-12 and IL-23 production by myeloid cells, thereby explaining predisposition to mycobacterial disease. Martin-Fernandez et al. describe patients with partial USP18 deficiency, which underlies both type I interferonopathy and Mendelian susceptibility to mycobacterial disease (MSMD). This work delineates the lack of negative regulation of the IFN-I signaling pathway leading to depression of the IFN-gamma-IL12 loop as a cause of MSMD.