Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.

SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

Nsp3s are the largest nonstructural proteins of coronaviruses. These transmembrane proteins include papain-like proteases (PLpro) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro of SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if the deubiquitinating activity of SARS-CoV-2 PLpro affects proteins that are substrates for ER-associated degradation (ERAD). Using full-length Nsp3 as well as a truncated transmembrane form we interrogated, by coexpression, three potential ERAD substrates, all of which play roles in regulating lipid biosynthesis. Transmembrane PLpro increases the level of INSIG-1 and decreases its ubiquitination. However, different effects were seen with SREBP-1 and SREBP-2. Transmembrane PLpro cleaves SREBP-1 at three sites, including two noncanonical sites in the N-terminal half of the protein, resulting in a decrease in precursors of the active transcription factor. Conversely, cleavage of SREBP-2 occurs at a single canonical site that disrupts a C-terminal degron, resulting in increased SREBP-2 levels. When this site is mutated and the degron can no longer be interrupted, SREBP-2 is still stabilized by transmembrane PLpro, which correlates with a decrease in SREBP-2 ubiquitination. All of these observations are dependent on PLpro catalytic activity. Our findings demonstrate that, when anchored to the ER membrane, SARS-CoV-2 Nsp3 PLpro can function as a deubiquitinating enzyme to stabilize ERAD substrates. Additionally, SARS-CoV-2 Nsp3 PLpro can cleave ER-resident proteins, including at sites that could escape analyses based on the established consensus sequence.

The α2,8-sialyltransferase 6 (St8sia6) localizes in the ER and enhances the anchorage-independent cell growth in cancer.

Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.

Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.

HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N6-methyladenosine (m6A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with liver-specific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m6A modification pathways, termed the ERm6A pathway, for ER stress adaptation to proteotoxicity.

P5A ATPase controls ER translocation of Wnt in neuronal migration.

The Wnt family contains conserved secretory proteins required for developmental patterning and tissue homeostasis. However, how Wnt is targeted to the endoplasmic reticulum (ER) for processing and secretion remains poorly understood. Here, we report that CATP-8/P5A ATPase directs neuronal migration non-cell autonomously in Caenorhabditis elegans by regulating EGL-20/Wnt biogenesis. CATP-8 likely functions as a translocase to translocate nascent EGL-20/Wnt polypeptide into the ER by interacting with the highly hydrophobic core region of EGL-20 signal sequence. Such regulation of Wnt biogenesis by P5A ATPase is common in C. elegans and conserved in human cells. These findings describe the physiological roles of P5A ATPase in neural development and identify Wnt proteins as direct substrates of P5A ATPase for ER translocation.

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Silencing of ER-resident oxidoreductase PDIA3 inhibits malignant biological behaviors of multidrug-resistant gastric cancer.

Glycosylation is a common posttranslational modification of proteins, which plays a role in the malignant transformation, growth, progression, chemoresistance, and immune response of tumors. Disulfide isomerase family A3 (PDIA3) specifically acts on newly synthesized glycoproteins to promote the correct folding of sugar chains. Studies have shown that PDIA3 participates in multidrug-resistant gastric cancer (MDR-GC). In this study, we performed western blot analysis and immunohistochemistry to identify PDIA3 expression. Cell proliferation was assessed by CCK-8 assay. Transwell assays were used to detect the migration and invasion abilities of cells. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis was employed to identify PDIA3-interacting proteins and the associated pathways in MDR-GC cells. Glycoprotein interactions and translocation were detected by immunofluorescence assay. The results showed that PDIA3 knockdown significantly inhibited the proliferation, invasion, and migration abilities of MDR-GC cells. Kyoto Encyclopedia of Genes and Genomes analysis of the IP-MS results showed that PDIA3 was closely associated with focal adhesion pathways in MDR-GC cells. Additionally, important components of focal adhesion pathways, including fibronectin-1 (FN1) and integrin α5 (ITGA5), were identified as pivotal PDIA3-binding glycoproteins. Knockdown of PDIA3 altered the cellular locations of FN1 and ITGA5, leading to abnormal accumulation. In conclusion, our results suggest that knockdown of PDIA3 inhibited the malignant behaviors of MDR-GC cells and influenced the translocation of FN1 and ITGA5.

Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage.

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.

Hypercapnia Induces Inositol-Requiring Enzyme 1α-Driven Endoplasmic Reticulum-associated Degradation of the Na,K-ATPase ß-Subunit.

Acute respiratory distress syndrome is often associated with elevated levels of CO2 (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA ß-subunit (NKA-ß) in the ER. Exposing murine precision-cut lung slices and human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid decrease of NKA-ß abundance in the ER and at the cell surface. Knockdown of ER mannosidase α class 1B member 1 and ER degradation-enhancing α-mannosidase like protein 1 by siRNA or treatment with the mannosidase α class 1B member 1 inhibitor kifunensine rescued loss of NKA-ß in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α), and treatment with an siRNA against IRE1α prevented the decrease of NKA-ß in the ER. Of note, the hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1α in precision-cut lung slices and A549 cells, suggesting that Ca2+ efflux from the ER might be responsible for IRE1α activation and ERAD of NKA-ß. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia.

Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT.

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

Neutrophils drive endoplasmic reticulum stress-mediated apoptosis in cancer cells through arginase-1 release.

Human neutrophils constitutively express high amounts of arginase-1, which depletes arginine from the surrounding medium and downregulates T-cell activation. Here, we have found that neutrophil arginase-1, released from activated human neutrophils or dead cells, induced apoptosis in cancer cells through an endoplasmic reticulum (ER) stress pathway. Silencing of PERK in cancer cells prevented the induction of ER stress and apoptosis. Arginase inhibitor Nω-hydroxy-nor-arginine inhibited apoptosis and ER stress response induced by conditioned medium from activated neutrophils. A number of tumor cell lines, derived from different tissues, were sensitive to neutrophil arginase-1, with pancreatic, breast, ovarian and lung cancer cells showing the highest sensitivity. Neutrophil-released arginase-1 and arginine deprivation potentiated the antitumor action against pancreatic cancer cells of the ER-targeted antitumor alkylphospholipid analog edelfosine. Our study demonstrates the involvement of neutrophil arginase-1 in cancer cell killing and highlights the importance and complex role of neutrophils in tumor surveillance and biology.

Inactivation of cysteine 674 in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 causes retinopathy in the mouse.

Diabetic retinopathy is a multifactorial microvascular complication, and its pathogenesis hasn't been fully elucidated. The irreversible oxidation of cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) was increased in the type 1 diabetic retinal vasculature. SERCA2 C674S knock-in (SKI) mouse line that half of C674 was replaced by serine 674 (S674) was used to study the effect of C674 inactivation on retinopathy. Compared with wild type (WT) mice, SKI mice had increased number of acellular capillaries and pericyte loss similar to those in type 1 diabetic WT mice. In the retina of SKI mice, pro-apoptotic proteins and intracellular Ca2+-dependent signaling pathways increased, while anti-apoptotic proteins and vessel density decreased. In endothelial cells, C674 inactivation increased the expression of pro-apoptotic proteins, damaged mitochondria, and induced cell apoptosis. These results suggest that a possible mechanism of retinopathy induced by type 1 diabetes is the interruption of calcium homeostasis in the retina by oxidation of C674. C674 is a key to maintain retinal health. Its inactivation can cause retinopathy similar to type 1 diabetes by promoting apoptosis. SERCA2 might be a potential target for the prevention and treatment of diabetic retinopathy.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

AIMS: Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. METHODS AND RESULTS: We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. CONCLUSIONS: We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

Enhanced triacylglycerol production in oleaginous microalga Neochloris oleoabundans by co-overexpression of lipogenic genes: Plastidial LPAAT1 and ER-located DGAT2.

Microalgae accumulate lipid triacylglycerol (TAG), a promising feedstock for production of natural edible oils and biofuels. To make products derived from microalgal TAG economically viable, increasing TAG content and productivity are of high importance. To increase TAG content, two endogenous key enzymes of TAG biosynthesis: plastidial lysophosphatidic acid acyltransferase (NeoLPAAT1) and endoplasmic reticulum-located diacylglycerol acyltransferase 2 (NeoDGAT2) were co-overexpressed in oleaginous microalga Neochloris oleoabundans. The neutral lipid content in NeoLPAAT1-NeoDGAT2 co-overexpressing transformant detected by Nile red staining increased 2-fold without compromising cell growth. The transcriptional levels of NeoLPAAT1 and NeoDGAT2 levels were 1.9-fold higher in the transformant than wild type. Considerably higher lipid accumulation was found in the transformant than wild type: total lipid content (73.72 ± 4.17 % DCW) increased 1.6-fold, TAG content (50.63 ± 6.15 % DCW) increased 2.1-fold, total lipid productivity (16.84 ± 0.66 mg/L/day) increased 1.9-fold, and TAG productivity (11.68 ± 0.90 mg/L/day) increased 2.1-fold. Fatty acid composition was slightly altered in the transformant compared to wild type; saturated fatty acid C16:0 increased to 26% from 20%, whereas C18:0 was reduced to 7% from 14%. Long-term stability of NeoLPAAT1-NeoDGAT2 co-overexpression was observed in the transformant continuously maintained on solid medium in a period of 4 years. The results suggested that targeted engineering of genes in pathway located at different organelles should be possible in microalgal lipid metabolism.

Misfolding, altered membrane distributions, and the unfolded protein response contribute to pathogenicity differences in Na,K-ATPase ATP1A3 mutations.

Missense mutations in ATP1A3, the α3 isoform of Na,K-ATPase, cause neurological phenotypes that differ greatly in symptoms and severity. A mechanistic basis for differences is lacking, but reduction of activity alone cannot explain them. Isogenic cell lines with endogenous α1 and inducible exogenous α3 were constructed to compare mutation properties. Na,K-ATPase is made in the endoplasmic reticulum (ER), but the glycan-free catalytic α subunit complexes with glycosylated ß subunit in the ER to proceed through Golgi and post-Golgi trafficking. We previously observed classic evidence of protein misfolding in mutations with severe phenotypes: differences in ER retention of endogenous ß1 subunit, impaired trafficking of α3, and cytopathology, suggesting that they misfold during biosynthesis. Here we tested two mutations associated with different phenotypes: D923N, which has a median age of onset of hypotonia or dystonia at 3 years, and L924P, with severe infantile epilepsy and profound impairment. Misfolding during biosynthesis in the ER activates the unfolded protein response, a multiarmed program that enhances protein folding capacity, and if that fails, triggers apoptosis. L924P showed more nascent protein retention in ER than D923N; more ER-associated degradation of α3 (ERAD); larger differences in Na,K-ATPase subunit distributions among subcellular fractions; and greater inactivation of eIF2α, a major defensive step of the unfolded protein response. In L924P there was also altered subcellular distribution of endogenous α1 subunit, analogous to a dominant negative effect. Both mutations showed pro-apoptotic sensitization by reduced phosphorylation of BAD. Encouragingly, however, 4-phenylbutyrate, a pharmacological corrector, reduced L924P ER retention, increased α3 expression, and restored morphology.

Variations in MHC class I antigen presentation and immunopeptidome selection pathways.

Major histocompatibility class I (MHC-I) proteins mediate immunosurveillance against pathogens and cancers by presenting antigenic or mutated peptides to antigen receptors of CD8+ T cells and by engaging receptors of natural killer (NK) cells. In humans, MHC-I molecules are highly polymorphic. MHC-I variations permit the display of thousands of distinct peptides at the cell surface. Recent mass spectrometric studies have revealed unique and shared characteristics of the peptidomes of individual MHC-I variants. The cell surface expression of MHC-I-peptide complexes requires the functions of many intracellular assembly factors, including the transporter associated with antigen presentation (TAP), tapasin, calreticulin, ERp57, TAP-binding protein related (TAPBPR), endoplasmic reticulum aminopeptidases (ERAPs), and the proteasomes. Recent studies provide important insights into the structural features of these factors that govern MHC-I assembly as well as the mechanisms underlying peptide exchange. Conformational sensing of MHC-I molecules mediates the quality control of intracellular MHC-I assembly and contributes to immune recognition by CD8 at the cell surface. Recent studies also show that several MHC-I variants can follow unconventional assembly routes to the cell surface, conferring selective immune advantages that can be exploited for immunotherapy.

ER-resident oxidoreductases are glycosylated and trafficked to the cell surface to promote matrix degradation by tumour cells.

Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.

The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase.

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

Noncanonical function of long myosin light chain kinase in increasing ER-PM junctions and augmentation of SOCE.

Increased endothelial permeability leads to excessive exudation of plasma proteins and leukocytes in the interstitium, which characterizes several vascular diseases including acute lung injury. The myosin light chain kinase long (MYLK-L) isoform is canonically known to regulate the endothelial permeability by phosphorylating myosin light chain (MLC-P). Compared to the short MYLK isoform, MYLK-L contains an additional stretch of ~919 amino acid at the N-terminus of unknown function. We show that thapsigargin and thrombin-induced SOCE was markedly reduced in Mylk-L-/- endothelial cells (EC) or MYLK-L-depleted human EC. These agonists also failed to increase endothelial permeability in MYLK-L-depleted EC and Mylk-L-/- lungs, thus demonstrating the novel role of MYLK-L-induced SOCE in increasing vascular permeability. MYLK-L augmented SOCE by increasing endoplasmic reticulum (ER)-plasma membrane (PM) junctions and STIM1 translocation to these junctions. Transduction of N-MYLK domain (amino acids 1-919 devoid of catalytic activity) into Mylk-L-/- EC rescued SOCE to the level seen in control EC in a STIM1-dependent manner. N-MYLK-induced SOCE augmented endothelial permeability without MLC-P via an actin-binding motif, DVRGLL. Liposomal-mediated delivery of N-MYLK mutant but not ∆DVRGLL-N-MYLK mutant in Mylk-L-/- mice rescued vascular permeability increase in response to endotoxin, indicating that targeting of DVRGLL motif within MYLK-L may limit SOCE-induced vascular hyperpermeability.