Biology subjects

Ginsburg, D.

Publications and source records attributed to Ginsburg, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Pathogenic MYBPC3 missense variants alter protein-protein interactions within the sarcomere

BackgroundHypertrophic cardiomyopathy (HCM) is a genetic heart disease that leads to left ventricular hypertrophy, heart failure, and arrhythmias. Pathogenic missense variants in the gene myosin binding protein C (MYBPC3) cluster within its internal subdomains C3 and C6. The protein (MyBP-C), carrying these missense variants, localizes normally to the myofilaments, leaving uncertainty regarding the mechanism(s) by which they cause HCM. MethodsWe probed the molecular pathogenesis of these variants by analyzing (1) their prevalence in an international registry of patients with HCM, (2) total MyBP-C levels and the allelic fraction of mutant MyBP-C in human left ventricular heart tissue, and (3) performing flag-immunoprecipitation and proximity labeling mass spectrometry of wild-type MyBP-C and four pathogenic missense variants (Arg495Gln, Arg502Trp- C3, Trp792Arg, Arg810His- C6) to determine the change in MyBP-C interacting and proximity proteins induced by these variants. ResultsWe found that among patients with HCM who had any MYBPC3 pathogenic variant, 17.9% had a missense variant within the C3 or C6 subdomain. Unlike truncating variants, these C3 or C6 missense variants did not reduce MyBP-C content relative to myosin. The mutant allelic fraction of MyBP-C varied from 10.0-67.0% across samples. Flag-immunoprecipitation mass spectrometry identified 252 MyBP-C interacting proteins. Pathogenic missense variants disrupted 23 MyBP-C protein interactions, including lysosomal Ragulator-Rag complex proteins (RRAGA, RRAGC, LAMTOR4). Proximity labeling mass spectrometry was more sensitive, identifying 3,240 MyBP-C proximity proteins. Pathogenic missense variant(s) altered the proximity to MyBP-C of 789 proteins (69.4% increased and 30.5% decreased). Proteins that were increased in proximity to the missense MyBP-C were enriched for thin-filament proteins. ConclusionPathogenic MYBPC3 missense variants within the C3 and C6 subdomains are present in a substantial subset of patients with HCM. Our findings imply unique mechanisms of these variants distinct from haploinsufficiency, potentially driven by enhanced proximity to the thin filament within myofilaments.

cell biology↗

bioRxiv2026

Mapping the Mutational Landscape for Streptokinase Binding to Plasminogen

Group A Streptococcus (GAS) expresses streptokinase (SK), a critical virulence factor that non-enzymatically activates the hosts plasminogen (PLG), to an active form, PLGSK, resulting in the degradation of fibrin clots and subsequent bacterial dissemination. PLGSK formation contrasts with the physiologic activation of PLG to the serine protease plasmin via a proteolytic mechanism. As a potent thrombolytic, SK has been used as a therapeutic to treat heart attacks and strokes. GAS SK is highly specific for human PLG, and sequence variation between SK from different GAS strains has been linked to differences in PLG binding and disease severity. We now report the application of deep mutational scanning (DMS) to map the effects of [~]71% of single amino acid substitutions within SK from Group C Streptococcus, which shares high sequence homology with GAS on its ability to bind human PLG. We first demonstrate that SK expressed as a fusion protein to the p3 coat protein of M13 filamentous phage retains its capacity to bind human PLG. Our subsequent DMS analysis using this phage system identifies regions of SK in which amino acid substitutions are likely to increase or decrease its affinity for PLG. Our findings suggest a complex protein-protein interaction in which long-range protein dynamics influence the conformational activation of PLG to PLGSK. These data lay the foundation for linking SK variation between GAS strains to differences in virulence, mapping the determinants of GAS SKs human specificity, and potentially contributing to the development of improved therapeutics for heart attack and stroke.

microbiology↗

bioRxiv2025

Thermodynamics and selection of the plasminogen activator inhibitor-1 latency transition

Plasminogen activator inhibitor-1 (PAI-1), like other serine protease inhibitors (SERPINs), exists in a functionally active metastable conformation. Unlike other SERPINs, PAI-1 undergoes a relatively rapid rearrangement to a lower energy latent conformation. We employ deep mutational scanning (DMS) to simultaneously probe the effects of nearly all single amino acid substitutions (92%) in PAI-1 on its latency transition as well as its ability to inhibit its canonical protease target, urokinase-type plasminogen activator (uPA). The DMS results are interpreted in the context of variant effect predictors (EVE, AlphaMissense, and CPT) and protein stability predictors (EvoEF and FoldX), as well as the evolutionary conservation of the PAI-1 sequence space across extant vertebrate species. We demonstrate that while variant effect predictors can generally partition PAI-1 as functional or non-functional with respect to uPA inhibition, they perform poorly when attempting to discriminate the effects of PAI-1 variants on its latency transition. However, by employing protein stability predictors, we demonstrate that the PAI-1 latency transition is most likely driven by changes in the energy barrier to the latency transition. Finally, we show that PAI-1s ability to inhibit uPA, as well as its thermodynamic instability in its active conformation are both under purifying selection that limits the sequence space available to PAI-1. These findings suggest that DMS of collateral fitness effects, including PAI-1s latency transition, may be better interpreted in contexts other than variant effect predictors, including protein thermodynamics and evolution.

biochemistry↗

bioRxiv2025

Characterizing venom resistance in Monodelphis domestica yields new insights into mammalian blood physiology

Interactions between predators and prey are often characterized by strong selection pressures that shape extreme physiological adaptations. Venom resistance in large-bodied South American opossums (Clade Didelphini) is a striking example, as these marsupials prey on venomous snakes and exhibit remarkable resistance to their venom. While resistance is well documented in Didelphini, relatively little is known about venom resistance in the smaller, more diverse members of Didelphidae, which inhabit the same regions and encounter the same predators. Here, we investigate venom resistance in the small-bodied opossum, Monodelphis domestica, through multi-level physiological assays, examining responses to purified venom components and whole venom from sympatric and allopatric vipers. Our results show M. domestica resists venom-induced disruptions to blood coagulation, retains platelet function in the presence of platelet-disrupting venoms, and inhibits snake venom metalloproteinases. Unexpectedly, we find that M. domestica von Willebrand Factor (VWF) requires increased shear force to elongate, a previously unknown aspect of opossum blood physiology that may contribute to venom resistance and may have relevance to human coagulopathies. These findings expand the extent of venom resistance beyond large-bodied Didelphini, suggesting it is a widespread trait in South American marsupials and providing new insights into venom-mammal coevolution. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/634112v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1efb74forg.highwire.dtl.DTLVardef@ebcb82org.highwire.dtl.DTLVardef@7ddef2org.highwire.dtl.DTLVardef@1b81569_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2025

Tandem duplication of serpin genes yields functional variation and snake venom inhibitors.

Tandem duplication of genes can play a critical role in the evolution of functional novelty, but our understanding is limited concerning gene duplications role in coevolution between species. Much is known about the evolution and function of tandemly duplicated snake venom genes, however the potential of gene duplication to fuel venom resistance within prey species is poorly understood. In this study, we characterize patterns of gene duplication of the SERPINA subfamily of genes across in vertebrates and experimentally characterize functional variation in the SERPINA3-like paralogs of a wild rodent. We find the hallmarks of rapid birth-death evolution of SERPINA1-like and SERPINA3-like genes within and between rodent lineages. Next, we recombinantly expressed the 2 paralogous duplicates of SERPINA1 and 12 paralogous duplicates of SERPINA3 found in the genome of the big-eared woodrat (Neotoma macrotis), a species known to be resistant to protease-rich rattlesnake venoms. We found that two SERPINA3 paralogs inhibit snake venom serine proteases, indicating that these proteins have potential as resistance factors in SERPIN-mediated venom resistance. In addition, functional variation is apparent among paralogs, including neofunctionalization to inhibit both chymotrypsin-like and and trypsin-like proteases simultaneously for one venom-inhibiting paralog. Our results provide further evidence that the rapid evolution of SERPINA1 and SERPINA3 gene copy number across rodents has adaptive potential by producing functionally-diverse inhibitors.

evolutionary biology↗

bioRxiv2024

Synergy of LPS and Shiga-toxin exposure on the RNA expression program of cultured human umbilical vein endothelial cells.

BackgroundHemolytic uremic syndrome (HUS) is a primary thrombotic microangiopathy that can cause acute renal failure, particularly in children, and is commonly triggered by Shiga toxin-producing Escherichia coli (STEC) infection. Although previous in vitro studies have shown that Shiga toxin (STX) can directly affect the endothelium, gram-negative bacteria such as E. coli also contain lipopolysaccharide (LPS) which is known to independently activate endothelial cells. Furthermore, studies in mice and baboons have shown a synergistic effect of LPS and STX in inducing HUS when administered simultaneously. ObjectiveDetermine how LPS, STX, or combined LPS/STX treatment affect the gene expression program of endothelial cells. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with STX, LPS or a combination of STX and LPS for either 4 or 24 hours, after which unbiased RNA sequencing analysis was performed. ResultsFour-hour treatment with LPS resulted in a greater number of differentially expressed genes as compared to treatment with STX. In contrast, 24-hour treatment with LPS or STX induced similar numbers of differentially expressed genes. Dual treatment with both toxins resulted in a synergistic effect, showing significant up-or downregulation of multiple transcripts that were not observed in either single treatment group, particularly at the 24-hour timepoint. ConclusionOur data provide a resource for assessing the role of endothelial cell gene expression in the pathogenesis of STEC infection and suggest that the responses of these cells to LPS, STX, or combined LPS/STX treatment are unique and temporally regulated.

physiology↗

bioRxiv2024

High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue-and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI-1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI-1 inhibitory function toward these non-canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of [~]80% of all possible single amino acid substitutions in PAI-1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI-1 mutational landscape, with the determinants of protease specificity distributed throughout PAI-1s primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI-1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection. PAI-1s activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of all secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.

evolutionary biology↗

bioRxiv2024

Functional overlap between the mammalian SAR1A and SAR1B paralogs in vivo

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit [~]90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during mid-embryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these 2 paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

genetics↗

bioRxiv2023

Identification of LMAN1 and SURF4 dependent secretory cargoes

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles/tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif.

cell biology↗

bioRxiv2022

Regulation of cellular LDL uptake by PROX1 and CHD7

An elevated level of low-density lipoprotein (LDL) in the bloodstream is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). The low-density lipoprotein receptor (LDLR) is a critical regulator of circulating LDL, and increasing LDLR activity is an effective therapeutic approach to reduce circulating LDL cholesterol levels. In this study, we characterize PROX1 and CHD7, two genes we previously identified in a genome-scale CRISPR screen as positive regulators of LDL uptake in HuH7 cells. We found that although disruption of either PROX1 or CHD7 significantly reduced LDL uptake, only PROX1 disruption significantly reduced the cellular levels of LDLR mRNA and surface-displayed LDLR protein. Consistent with a direct role for PROX1 in LDLR gene regulation, we also observed in publicly available data sets the presence of two liver-specific PROX1 binding sites near the LDLR locus, one of which colocalized with biochemical hallmarks of enhancer activity in hepatic tissue. Both PROX1 LDLR binding sites contained predicted PROX1 transcription factor binding motifs and colocalized with binding sites for HNF4, a known interactor for PROX1 and regulator of hepatic lipid metabolism and LDL uptake. In contrast to PROX1, no CHD7 binding sites were detected near the LDLR locus. Together, our results support a model in which both PROX1 and CHD7 promote cellular LDL uptake through distinct mechanisms, with PROX1 directly promoting LDLR gene expression and CHD7 functioning through an LDLR-independent pathway.

molecular biology↗

bioRxiv2022

Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by {bsim}60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding {bsim}50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B. Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.

physiology↗

bioRxiv2022

Deep mutational scanning and massively parallel kinetics of plasminogen activator-1 functional stability

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily of proteins, is unique among SERPINs for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ~1-2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 {degrees}C. Of the 7,201 possible single amino acid substitutions in PAI-1, we identify 439 that increase the functional stability of PAI-1 beyond that of the wild-type protein and 1,549 that retain inhibitory activity toward PAI-1s canonical target protease (urokinase-like plasminogen activator, uPA), while exhibiting functional stability less than or equal to that of wild-type PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying PAI-1s latency transition and provide a database for interpreting human PAI-1 genetic variants.

biochemistry↗

bioRxiv2022

Identification of secreted proteins by comparison of protein abundance in conditioned media and cell lysates.

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking.

biochemistry↗

bioRxiv2021

Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants may render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. The effects of single amino acid substitutions on PAI-1s functional inhibition of its canonical target proteases, tPA and uPA, have been determined for only a small fraction of potential mutations. To construct a more comprehensive dataset, a mutagenized PAI-1 library, encompassing [~]70% of potential single amino acid substitutions, was displayed on M13 filamentous phage. From this library, the relative effects of 27% of all possible missense variants on PAI-1 inhibition of urokinase-type plasminogen activator were determined using high-throughput DNA sequencing with 826 missense variants demonstrating conserved inhibitory activity and 1137 resulting in loss of PAI-1 function. Comparison of these deep mutational scanning results to predictions from PolyPhen-2 and SIFT demonstrate the limitations of these algorithms, consistent with similar reports for other proteins. Comparison to common human PAI-1 gene variants present in the gnomAD database is consistent with evolutionary selection against loss of PAI-1 function. These findings provide insight into structure-function relationships for PAI-1 and other members of the SERPIN superfamily.

biochemistry↗

bioRxiv2021

Phage Display Broadly Identifies Inhibitor-reactive Regions in von Willebrand Factor

BackgroundCorrection of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti-VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD). ObjectiveLocate inhibitor-reactive regions within VWF using phage display. MethodsWe screened a phage library displaying random, overlapping fragments covering the full length VWF protein sequence for binding to a commercial anti-VWF antibody or to immunoglobulins from three type 3 VWD patients who developed VWF inhibitors in response to treatment with plasma-derived VWF. Immunoreactive phage clones were identified and quantified by next generation DNA sequencing (NGS). ResultsNGS markedly increased the number of phage analyzed for locating immunoreactive regions within VWF following a single round of selection and identified regions not recognized in previous reports using standard phage display methods. Extending this approach to characterize VWF inhibitors from three type 3 VWD patients (including two siblings homozygous for the same VWF gene deletion) revealed patterns of immunoreactivity distinct from the commercial antibody and between unrelated patients, though with notable areas of overlap. Alloantibody reactivity against the VWF propeptide is consistent with incomplete removal of the propeptide from plasma-derived VWF replacement products. ConclusionThese results demonstrate the utility of phage display and NGS to characterize diverse anti-VWF antibody reactivities.

pathology↗

bioRxiv2020

Genome-scale CRISPR screening for modifiers of cellular LDL uptake

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.

genomics↗

bioRxiv2019

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression following EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single cell suspensions for fluorescence-activated cell sorting (FACS) or single cell RNA sequencing analysis. Comparison of our EC-TRAP to published single cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.\n\nSignificanceEndothelial cells (ECs), which line all vertebrate blood vessels, are highly heterogeneous across different tissues. The present study uses a genetic approach to specifically tag mRNAs within ECs of the mouse, thereby allowing recovery and sequence analysis to evaluate the EC-specific gene expression program directly from intact organs. Our findings demonstrate marked heterogeneity in EC gene expression across different vascular beds under both normal and disease conditions, with a more accurate picture than can be achieved using other methods. The data generated in these studies advance our understanding of EC function in different blood vessels and provide a valuable resource for future studies.

genomics↗

bioRxiv2019

Murine Surf4 is essential for early embryonic development

Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4. Surf4+/- mice exhibited grossly normal appearance, behavior, body weight, fecundity, and organ development and demonstrated no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol. Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. Taken together with the much milder phenotypes of PCSK9 or apolipoprotein B deficiency in mice, these findings imply the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.

cell biology↗