Golgi Reassembly Stacking Protein Research Articles

Discovery of Cell Number-Interstitial Fluid Volume (CIF) Ratio Reveals Secretory Autophagy Pathway to Supply eHsp90α for Wound Healing

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately <2%, <4% and >95%, respectively. Establishing the cell-number-to-interstitial-fluid-volume (CIF) ratio for the microenvironment of human tissues as 1 × 109 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 μg/mL, 5.61 μg/mL and 178 μg/mL. Remarkably, the 178 μg/mL eHsp90α matches the previously reported 100–300 μg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data.

Golgi defect as a major contributor to lysosomal dysfunction.

The Golgi apparatus plays a crucial role in lysosome biogenesis and the delivery of lysosomal enzymes, essential for maintaining cellular homeostasis and ensuring cell survival. Deficiencies in Golgi structure and function can profoundly impact lysosomal homeostasis, leading to various lysosomal storage diseases and neurodegenerative disorders. In this review, we highlight the role of the Golgi Reassembly Stacking Proteins (GRASPs) in the formation and function of the Golgi apparatus, emphasizing the current understanding of the association between the Golgi apparatus, lysosomes, and lysosomal storage diseases. Additionally, we discuss how Golgi dysfunction leads to the secretion of lysosomal enzymes. This review aims to serve as a concise resource, offering insights into Golgi structure, function, disease-related defects, and their consequential effects on lysosomal biogenesis and function. By highlighting Golgi defects as an underappreciated contributor to lysosomal dysfunction across various diseases, we aim to enhance comprehension of these intricate cellular processes.

Golgi Reassembly Stacking Protein 2 Modulates Myometrial Contractility during Labor by Affecting ATP Production.

The mechanism of maintaining myometrial contractions during labor remains unclear. Autophagy has been reported to be activated in laboring myometrium, along with the high expression of Golgi reassembly stacking protein 2 (GORASP2), a protein capable of regulating autophagy activation. This study aimed to investigate the role and mechanism of GORASP2 in uterine contractions during labor. Western blot confirmed the increased expression of GORASP2 in laboring myometrium. Furthermore, the knockdown of GORASP2 in primary human myometrial smooth muscle cells (hMSMCs) using siRNA resulted in reduced cell contractility. This phenomenon was independent of the contraction-associated protein and autophagy. Differential mRNAs were analyzed using RNA sequencing. Subsequently, KEGG pathway analysis identified that GORASP2 knockdown suppressed several energy metabolism pathways. Furthermore, reduced ATP levels and aerobic respiration impairment were observed in measuring the oxygen consumption rate (OCR). These findings suggest that GORASP2 is up-regulated in the myometrium during labor and modulates myometrial contractility mainly by maintaining ATP production.

Golgi's Role in the Development of Possible New Therapies in Cancer.

The Golgi apparatus is an important organelle found in most eukaryotic cells. It plays a vital role in the processing and sorting of proteins, lipids and other cellular components for delivery to their appropriate destinations within the cell or for secretion outside of the cell. The Golgi complex also plays a role in the regulation of protein trafficking, secretion and post-translational modifications, which are significant in the development and progression of cancer. Abnormalities in this organelle have been observed in various types of cancer, although research into chemotherapies that target the Golgi apparatus is still in its early stages. There are a few promising approaches that are being investigated: (1) Targeting the stimulator of interferon genes protein: The STING pathway senses cytosolic DNA and activates several signaling events. It is regulated by numerous post-translational modifications and relies heavily on vesicular trafficking. Based on some observations which state that a decreased STING expression is present in some cancer cells, agonists for the STING pathway have been developed and are currently being tested in clinical trials, showing encouraging results. (2) Targeting glycosylation: Altered glycosylation, which refers to changes in the carbohydrate molecules that are attached to proteins and lipids in cells, is a common feature of cancer cells, and there are several methods that thwart this process. For example, some inhibitors of glycosylation enzymes have been shown to reduce tumor growth and metastasis in preclinical models of cancer. (3) Targeting Golgi trafficking: The Golgi apparatus is responsible for the sorting and trafficking of proteins within the cell, and disrupting this process may be a potential therapeutic approach for cancer. The unconventional protein secretion is a process that occurs in response to stress and does not require the involvement of the Golgi organelles. P53 is the most frequently altered gene in cancer, dysregulating the normal cellular response to DNA damage. The mutant p53 drives indirectly the upregulation of the Golgi reassembly-stacking protein 55kDa (GRASP55). Through the inhibition of this protein in preclinical models, the reduction of the tumoral growth and metastatic capacity have been obtained successfully. This review supports the hypothesis that the Golgi apparatus may be the target of cytostatic treatment, considering its role in the molecular mechanisms of the neoplastic cells.

Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction.

Stable cell lines that express a gene of specific interest provide an advantage over transient gene expression by reducing variations in transfection efficiency between experiments, sustaining expression for long-term studies, and controlling expression levels in particular if a clonal population is selected. Transient transfection requires introduction of an exogenous gene into host cells via typically harsh chemicals or conditions that permeabilize the cell membrane, which does not normally integrate into the target cell genome. Here, we describe the method of using retroviral transduction to stably express Golgi proteins fused to a promiscuous biotin ligase (TurboID) in HeLa cells, thus creating cell lines that can be leveraged in studies of the proximome/interactome. We also demonstrate a similar protocol for stable expression of a Golgi protein fused to a fluorescent tag via lentiviral transduction. These methods can be further adapted to establish other cell lines with different sub-cellular markers or fusion tags. Viral transduction is a convenient method to create stable cell lines in cell-based studies.

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.

GRASP55 restricts early-stage autophagy and regulates spatial organization of the early secretory network.

ABSTRACTThere is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)­­-Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

Cu/Zn superoxide dismutase (VdSOD1) mediates reactive oxygen species detoxification and modulates virulence in Verticillium dahliae.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn‐SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione‐treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more O2‐ and less H2O2 than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host‐generated extracellular ROS, and contributes significantly to virulence in V. dahliae.

PsGRASP, a Golgi Reassembly Stacking Protein in Phytophthora sojae, Is Required for Mycelial Growth, Stress Responses, and Plant Infection.

Golgi reassembly stacking proteins (GRASPs) play important roles in Golgi structure formation, ER stress response, and unconventional secretion in eukaryotic cells. However, GRASP functions in oomycetes haven’t been adequately characterized. Here, we report the identification and functional analysis of PsGRASP, a GRASP-encoding gene from the soybean-infecting oomycete Phytophthora sojae. Transcriptional profiling showed that PsGRASP expression is up-regulated at the infection stages. PsGRASP knockout mutants were created using the CRISPR/Cas9 system. These mutants exhibited impaired vegetative growth, zoospore release and virulence. PsGRASP was involved ER stress responses and altered laccase activity. Our work suggests that PsGRASP is crucial for P. sojae development and pathogenicity.

The application of artificial intelligence methods to gene expression data for differentiation of uncomplicated and complicated appendicitis in children and adolescents - a proof of concept study \u2013

BackgroundGenome wide gene expression analysis has revealed hints for independent immunological pathways underlying the pathophysiologies of phlegmonous (PA) and gangrenous appendicitis (GA). Methods of artificial intelligence (AI) have successfully been applied to routine laboratory and sonographic parameters for differentiation of the inflammatory manifestations. In this study we aimed to apply AI methods to gene expression data to provide evidence for feasibility.MethodsModern algorithms from AI were applied to 56.666 gene expression data sets from 13 patients with PA and 16 with GA aged 7–17 years by using resampling methods (bootstrap). Performance with respect to sensitivities and specificities where investigated with receiver operating characteristic (ROC) analysis.ResultsWithin the experimental setting a best performing discriminatory biomarker signature consisting of a set of 4 genes could be defined: ERGIC and golgi 3, regulator of G-protein signaling 2, Rho GTPase activating protein 33, and Golgi Reassembly Stacking Protein 2. ROC analysis showed a mean area under the curve of 84%.ConclusionsGene expression based application of AI methods is feasible and represents a promising approach for future discriminatory diagnostics in children with acute appendicitis.

Amyloid beta regulates ER exit sites formation through O-GlcNAcylation triggered by disrupted calcium homeostasis.

Aberrant production of amyloid beta (Aβ) causes disruption of intracellular calcium homeostasis, a crucial factor in the pathogenesis of Alzheimer's disease. Calcium is required for the fusion and trafficking of vesicles. Previously, we demonstrated that Sec31A, a main component for coat protein complex II (COPII) vesicles at ER exit sites (ERES), is modulated by O-GlcNAcylation. O-GlcNAcylation, a unique and dynamic protein glycosylation process, modulates the formation of COPII vesicles. In this study, we observed that disrupted calcium levels affected the formation of COPII vesicles in ERES through calcium-triggered O-GlcNAcylation of Sec31A. Additionally, we found that Aβ impaired ERES through Aβ-disturbed calcium homeostasis and O-GlcNAcylation of Sec31A in neuronal cells. Furthermore, we identified that Aβ disrupted the ribbon-like structure of Golgi. Golgi fragmentation by Aβ was rescued by up-regulation of O-GlcNAcylaion levels using Thiamet G (ThiG), an O-GlcNAcase inhibitor. Additionally, we observed that the Golgi reassembly stacking proteins having a function in Golgi stacking showed attenuation at COPII vesicles following Aβ treatment. This study demonstrated that Aβ impaired Sec31A targeting to ERES through altered Sec31A O-GlcNAcylation triggered by disruption of intracellular calcium homeostasis. The findings of this study suggested that protection of ERES or Sec31 O-GlcNAcylation may offer a promising novel avenue for development of AD therapeutics.

Secretory autophagy machinery and vesicular trafficking are involved in HMGB1 secretion

ABSTRACT Nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern. Extracellular HMGB1 induces inflammation, cytokine production, and immune cell recruitment via activation of various receptors. As HMGB1 does not contain an endoplasmic reticulum-targeting signal peptide, HMGB1 is secreted via the endoplasmic reticulum-Golgi independently via an unconventional secretion pathway. However, the mechanism underlying HMGB1 secretion remains largely unknown. Here, we investigated the role of secretory autophagy machinery and vesicular trafficking in HMGB1 secretion. We observed that HSP90AA1 (heat shock protein 90 alpha family class A member 1), a stress-inducible protein, regulates the translocation of HMGB1 from the nucleus to the cytoplasm and its secretion through direct interaction. Additionally, geldanamycin, an HSP90AA1 inhibitor, reduced HMGB1 secretion. GORASP2/GRASP55 (golgi reassembly stacking protein 2), ARF1Q71L (ADP ribosylation factor 1), and SAR1AT39N (secretion associated Ras related GTPase 1A), which promoted unconventional protein secretion, increased HMGB1 secretion. HMGB1 secretion was inhibited by an early autophagy inhibitor and diminished in ATG5-deficient cells even when GORASP2 was overexpressed. In contrast, a late autophagy inhibitor increased HMGB1 secretion under the same conditions. The multivesicular body formation inhibitor GW4869 dramatically decreased HMGB1 secretion under HMGB1 secretion-inducing conditions. Thus, we demonstrated that secretory autophagy and multivesicular body formation mediate HMGB1 secretion.

Investigating the mechanisms of BK polyomavirus egress and virus-host interactions

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70-90% of the world's population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop a haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV associated nephropathy. While the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, we have evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. We have investigated the effects of knocking out cellular genes thought to be involved in unconventional secretory pathways that bypass the Golgi apparatus on the release of BKPyV. We observe decreased BKPyV release from cells that have undergone CRISPR-mediated knockout of Golgi Reassembly Stacking Protein (GORASP) 1 or 2. Investigation of BKPyV-induced changes to the plasma membrane of infected cells demonstrated increased cell surface expression of transmembrane proteins normally resident in the endoplasmic reticulum. This appears to be inhibited by the knockout of GORASP1 or 2, suggesting that virions and ER markers are secreted via a common pathway in infected cells. These experiments are uncovering novel virus-host interactions that, when targeted, could help prevent BKPyV-associated nephropathy and allograft loss.

Markers of malignant prostate cancer cells: Golgi localization of α-mannosidase 1A at GM130-GRASP65 site and appearance of high mannose N-glycans on cell surface

The ability to distinguish malignant from indolent prostate cancer cells is critically important for identification of clinically significant prostate cancer to minimize unnecessary overtreatment and sufferings endured by patients who have indolent cancer. Recently, we discovered that loss of giantin function as the primary Golgi targeting site for endoplasmic reticulum-derived transport vesicles in aggressive prostate cancer cells caused a shift of the Golgi localization site of α-mannosidase 1A to 130 KDa Golgi matrix protein (GM130)-65 KDa Golgi reassembly-stacking protein (GRASP65) site resulting in emergence of high mannose N-glycans on trans-Golgi enzymes and cell surface glycoproteins. To extend this observation, we isolated two cell clones (Clone 1 and Clone 2) from high passage LNCaP cells, which exhibited androgen refractory property missing in low passage LNCaP cells, and characterized their malignant property. We have found that comparing to Clone 2, which does not have cell surface high mannose N-glycans and exhibits localization of α-mannosidase 1A at giantin site, Clone 1 displays cell surface high mannose N-glycans, exhibits localization of α-mannosidase 1A at GM130-GRASP65 site, and shows a faster rate of closing the wound in a wound healing assay. The results indicate that Golgi localization of α-mannosidase 1A at GM130-GRASP65 site and appearance of cell surface high mannose N-glycans may serve as markers of malignant prostate cancer cells.

Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

ABSTRACT The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; GORASP2 shRNA: lentivirus harboring shRNA against GORASP2; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; HMGB1 shRNA: lentivirus harboring shRNA against HMGB1; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; Krt14+/+-Atg5f/f mice: mice bearing an Atg5 flox allele, in which exon 3 of the Atg5 gene is flanked by two loxP sites; Krt14+/+-Hmgb1f/f : mice bearing an Hmgb1 flox allele, in which exon 2 to 4 of the Hmgb1 gene is flanked by two loxP sites; Krt14Cre/+-atg5f/f mice: keratinocyte-specific atg5 knockout mice generated by mating Atg5-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt4; Krt14Cre/+-hmgb1f/f mice: keratinocyte-specific hmgb1 knockout mice generated by mating Hmgb1-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt14; Krt14-Vegfa mice: mice expressing 164-amino acid Vegfa splice variant recombinase under the control of promoter of Krt14; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; tcrd KO mice: tcrd (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.

The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII

BackgroundCongenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein.ResultsThrough screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures.ConclusionsThe present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion.

When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)-dependent but was not associated with ER stress. These data indicate that Hck-activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom-dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.

SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly.

Sirtuin 2 (SIRT2) is an NAD-dependent sirtuin deacetylase that regulates microtubule and chromatin dynamics, gene expression and cell cycle progression, as well as nuclear envelope reassembly. Recent proteomic analyses have identified Golgi proteins as SIRT2 interactors, indicating that SIRT2 may also play a role in Golgi structure formation. Here, we show that SIRT2 depletion causes Golgi fragmentation and impairs Golgi reassembly at the end of mitosis. SIRT2 interacts with the Golgi reassembly stacking protein GRASP55 (also known as GORASP2) in mitosis when GRASP55 is highly acetylated on K50. Expression of wild-type and the K50R acetylation-deficient mutant of GRASP55, but not the K50Q acetylation-mimetic mutant, in GRASP55 and GRASP65 (also known as GORASP1) double-knockout cells, rescued the Golgi structure and post-mitotic Golgi reassembly. Acetylation-deficient GRASP55 exhibited a higher self-interaction efficiency, a property required for Golgi structure formation. These results demonstrate that SIRT2 regulates Golgi structure by modulating GRASP55 acetylation levels.

DjA1 maintains Golgi integrity via interaction with GRASP65.

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

Brugada syndrome trafficking-defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels.

Cardiac Nav1.5 and Kir2.1-2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems - rat ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) - demonstrated that endoplasmic reticulum (ER) trafficking-defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking-defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking-defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.