Biology subjects

Ginsburg, D.

Publications and source records attributed to Ginsburg, D..

5 recordsLinked to original sources

bioRxiv2018

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intracellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extracellular secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient cellular secretion of PCSK9.

cell biology↗

bioRxiv2018

Murine SEC24D Can Substitute Functionally for SEC24C in vivo

The COPII component SEC24 mediates the recruitment of transmembrane cargoes or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E 7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no obvious developmental abnormality. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo function explain the early embryonic requirements for SEC24C and SEC24D.

cell biology↗

bioRxiv2017

The Function of the COPII Gene Paralogs SEC23A and SEC23B Are Interchangeable In Vivo

SEC23 is a core component of the coat protein-complex II (COPII)-coated vesicle, which mediates transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi1-3. Mammals express 2 paralogs for SEC23 (SEC23A and SEC23B). Though the SEC23 gene duplication dates back >500 million years, both SEC23s are ~85% identical at the amino acid sequence level. In humans, deficiency for SEC23A or SEC23B results in cranio-lenticulo-sutural dysplasia4 or congenital dyserythropoietic anemia type II (CDAII), respectively5. The disparate human syndromes and reports of secretory cargos with apparent paralog-specific dependence6,7, suggest unique functions for the two SEC23 paralogs. Here we show indistinguishable intracellular interactomes for human SEC23A and SEC23B, complementation of yeast SEC23 by both human and murine SEC23A/B paralogs, and the rescue of lethality resulting from Sec23b disruption in zebrafish by a Sec23a-expressing transgene. Finally, we demonstrate that the Sec23a coding sequence inserted into the endogenous murine Sec23b locus fully rescues the mortality and severe pancreatic phenotype previously reported with SEC23B-deficiency in the mouse8-10. Taken together, these data indicate that the disparate phenotypes of SEC23A and SEC23B deficiency likely result from evolutionary shifts in gene expression program rather than differences in protein function, a paradigm likely applicable to other sets of paralogous genes. These findings also suggest the potential for increased expression of SEC23A as a novel therapeutic approach to the treatment of CDAII, with potential relevance to other disorders due to mutations in paralogous genes.

evolutionary biology↗

bioRxiv2017

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ( rescues) using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p=0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p=0.01).\n\nAuthor summaryAbnormal blood clotting in veins (venous thrombosis) or arteries (arterial thrombosis) are major health problems, with venous thrombosis affecting approximately 1 in every thousand individuals annually in the United States. Susceptibility to venous thrombosis is governed by both genes and environment, with approximately 60% of the risk attributed to genetic influences. Though several genetic risk factors are known, >50% of genetic risk remains unexplained. Approximately 5% of people carry the most common known risk factor, Factor V Leiden. However, only 10% of these individuals will develop a blood clot in their lifetime. Mice carrying two copies of the Factor V Leiden mutation together with a mutation in a second gene called tissue factor pathway inhibitor develop fatal thrombosis shortly after birth. To identify genes that prevent this fatal thrombosis, we studied a large panel of mice carrying inactivating gene changes randomly distributed throughout the genome. We identified several genes as potential candidates to alter blood clotting balance in mice and humans with predisposition to thrombosis, and confirmed this protective function for DNA changes in one of these genes (Plcb4).

genetics↗

bioRxiv2016

A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci

Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-). F8 deficiency enhanced survival of F5L/L Tfpi+/- mice, demonstrating that F5L/L Tfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+ Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/L Tfpi+/- offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to as MF5L (Modifier of Factor 5 Leiden) 1-16. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/-) suppressed F5L/L Tfpi+/- lethality. Whole exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/L Tfpi+/- lethality (p=1.7x10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action for Actr2p.R258G. Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a novel role for Actr2 in this process.\n\nSignificance StatementVenous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ~10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.

genetics↗