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 midembryogenesis. 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 two 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.

Identification of cell type specific ACE2 modifiers by CRISPR screening.

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.

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 and 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..

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.

Genotype-Dependent and -Independent Calcium Signaling Dysregulation in Human Hypertrophic Cardiomyopathy.

BACKGROUND: Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. METHODS: We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). RESULTS: Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. CONCLUSIONS: Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.

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.

Increased endothelial sclerostin caused by elevated DSCAM mediates multiple trisomy 21 phenotypes.

Trisomy 21 (T21), a recurrent aneuploidy occurring in 1:800 births, predisposes to congenital heart disease (CHD) and multiple extracardiac phenotypes. Despite a definitive genetic etiology, the mechanisms by which T21 perturbs development and homeostasis remain poorly understood. We compared the transcriptome of CHD tissues from 49 patients with T21 and 226 with euploid CHD (eCHD). We resolved cell lineages that misexpressed T21 transcripts by cardiac single-nucleus RNA sequencing and RNA in situ hybridization. Compared with eCHD samples, T21 samples had increased chr21 gene expression; 11-fold-greater levels (P = 1.2 × 10-8) of SOST (chr17), encoding the Wnt inhibitor sclerostin; and 1.4-fold-higher levels (P = 8.7 × 10-8) of the SOST transcriptional activator ZNF467 (chr7). Euploid and T21 cardiac endothelial cells coexpressed SOST and ZNF467; however, T21 endothelial cells expressed 6.9-fold more SOST than euploid endothelial cells (P = 2.7 × 10-27). Wnt pathway genes were downregulated in T21 endothelial cells. Expression of DSCAM, residing within the chr21 CHD critical region, correlated with SOST (P = 1.9 × 10-5) and ZNF467 (P = 2.9 × 10-4). Deletion of DSCAM from T21 endothelial cells derived from human induced pluripotent stem cells diminished sclerostin secretion. As Wnt signaling is critical for atrioventricular canal formation, bone health, and pulmonary vascular homeostasis, we concluded that T21-mediated increased sclerostin levels would inappropriately inhibit Wnt activities and promote Down syndrome phenotypes. These findings imply therapeutic potential for anti-sclerostin antibodies in T21.

Cargo selection in endoplasmic reticulum-to-Golgi transport and relevant diseases.

Most proteins destined for the extracellular space or various intracellular compartments must traverse the intracellular secretory pathway. The first step is the recruitment and transport of cargoes from the endoplasmic reticulum (ER) lumen to the Golgi apparatus by coat protein complex II (COPII), consisting of five core proteins. Additional ER transmembrane proteins that aid cargo recruitment are referred to as cargo receptors. Gene duplication events have resulted in multiple COPII paralogs present in the mammalian genome. Here, we review the functions of each COPII protein, human disorders associated with each paralog, and evidence for functional conservation between paralogs. We also provide a summary of current knowledge regarding two prototypical cargo receptors in mammals, LMAN1 and SURF4, and their roles in human health and disease.

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.

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 ~60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding ~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 (APOB). 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.

Identification of ACE2 modifiers by CRISPR screening.

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1 , or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.

Myofibrillar Structural Variability Underlies Contractile Function in Stem Cell-Derived Cardiomyocytes.

Disease modeling and pharmaceutical testing using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) requires accurate assessment of contractile function. Micropatterning iPSC-CMs on elastic substrates controls cell shape and alignment to enable contractile studies, but determinants of intrinsic variability in this system have been incompletely characterized. The objective of this study was to determine the impact of myofibrillar structure on contractile function in iPSC-CMs. Automated analysis of micropatterned iPSC-CMs labeled with a cell-permeant F-actin dye revealed that myofibrillar abundance is widely variable among iPSC-CMs and strongly correlates with contractile function. This variability is not reduced by subcloning from single iPSCs and is independent of the iPSC-CM purification method. Controlling for myofibrillar structure reduces false-positive findings related to batch effect and improves sensitivity for pharmacologic testing and disease modeling. This analysis provides compelling evidence that myofibrillar structure should be assessed concurrently in studies investigating contractile function in iPSC-CMs.

Murine SEC24D can substitute functionally for SEC24C during embryonic development.

The COPII component SEC24 mediates the recruitment of transmembrane cargos 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 (E7.5) 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 other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.

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. Heterozygous Surf4+/- mice exhibit grossly normal appearance, behavior, body weight, fecundity, and organ development, with no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol, and a detectable accumulation of intrahepatic apoliprotein B. Homozygous Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. In contrast to the milder murine phenotypes associated with deficiency of known SURF4 cargoes, the embryonic lethality of Surf4-/- mice implies the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.

Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy.

Mutations in cardiac myosin binding protein C (MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy (HCM). Most MYBPC3 mutations result in premature termination codons (PTCs) that cause RNA degradation and a reduction of MyBP-C in HCM patient hearts. However, a reduction in MyBP-C has not been consistently observed in MYBPC3-mutant induced pluripotent stem cell cardiomyocytes (iPSCMs). To determine early MYBPC3 mutation effects, we used patient and genome-engineered iPSCMs. iPSCMs with frameshift mutations were compared with iPSCMs with MYBPC3 promoter and translational start site deletions, revealing that allelic loss of function is the primary inciting consequence of mutations causing PTCs. Despite a reduction in wild-type mRNA in all heterozygous iPSCMs, no reduction in MyBP-C protein was observed, indicating protein-level compensation through what we believe is a previously uncharacterized mechanism. Although homozygous mutant iPSCMs exhibited contractile dysregulation, heterozygous mutant iPSCMs had normal contractile function in the context of compensated MyBP-C levels. Agnostic RNA-Seq analysis revealed differential expression in genes involved in protein folding as the only dysregulated gene set. To determine how MYBPC3-mutant iPSCMs achieve compensated MyBP-C levels, sarcomeric protein synthesis and degradation were measured with stable isotope labeling. Heterozygous mutant iPSCMs showed reduced MyBP-C synthesis rates but a slower rate of MyBP-C degradation. These findings indicate that cardiomyocytes have an innate capacity to attain normal MyBP-C stoichiometry despite MYBPC3 allelic loss of function due to truncating mutations. Modulating MyBP-C degradation to maintain MyBP-C protein levels may be a novel treatment approach upstream of contractile dysfunction for HCM.

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9.

PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. 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. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.