Neurodegenerative diseases associated with the disruption of proteostasis and their therapeutic strategies using chemical chaperones.

Aberrant proteostasis is thought to be involved in the pathogenesis of neurodegenerative diseases. Some proteostasis abnormalities are ameliorated by chaperones. Chaperones are divided into three groups: molecular, pharmacological, and chemical. Chemical chaperones intended to alleviate stress in organelles, such as the endoplasmic reticulum (ER), are now being administered clinically. Of the chemical chaperones, 4-phenylbutyrate (4-PBA) has been used as a research reagent, and its mechanism of action includes chaperone effects and the inhibition of histone deacetylase. Moreover, it also binds to the B-site of SEC24 and regulates COPII-mediated transport from the ER. Although its therapeutic effect may not be strong, elucidating the mechanism of action of 4-PBA may contribute to the identification of novel therapeutic targets for neurodegenerative diseases.

Identification and characterization of the COPII vesicle-forming GTPase Sar1 in Chlamydomonas.

Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane-bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII-mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site-directed mutagenesis to generate a dominant-negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER-Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER-to-Golgi protein secretory pathway in green algae.

High levels of intracellular endotrophin in adipocytes mediate COPII vesicle supplies to autophagosome to impair autophagic flux and contribute to systemic insulin resistance in obesity.

BACKGROUND AND AIMS: Extracellular matrix (ECM) homeostasis plays a crucial role in metabolic plasticity and endocrine function of adipose tissue. High levels of intracellular endotrophin, a cleavage peptide of type VI collagen alpha 3 chain (Col6a3), have been frequently observed in adipocyte in obesity and diabetes. However, how endotrophin intracellularly traffics and influences metabolic homeostasis in adipocyte remains unknown. Therefore, we aimed to investigate the trafficking of endotrophin and its metabolic effects in adipocytes depending on lean or obese condition. METHODS: We used doxycycline-inducible adipocyte-specific endotrophin overexpressed mice for a gain-of-function study and CRISPR-Cas9 system-based Col6a3-deficient mice for a loss-of-function study. Various molecular and biochemical techniques were employed to examine the effects of endotrophin on metabolic parameters. RESULTS: In adipocytes during obesity, the majority of endosomal endotrophin escapes lysosomal degradation and is released into the cytosol to mediate direct interactions between SEC13, a major component of coat protein complex II (COPII) vesicles, and autophagy-related 7 (ATG7), leading to the increased formation of autophagosomes. Autophagosome accumulation disrupts the balance of autophagic flux, resulting in adipocyte death, inflammation, and insulin resistance. These adverse metabolic effects were ameliorated by either suppressing ATG7 with siRNA ex vivo or neutralizing endotrophin with monoclonal antibodies in vivo. CONCLUSIONS: High levels of intracellular endotrophin-mediated autophagic flux impairment in adipocyte contribute to metabolic dysfunction such as apoptosis, inflammation, and insulin resistance in obesity.

The C-terminus of the cargo receptor Erv14 affects COPII vesicle formation and cargo delivery.

The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. The Saccharomyces cerevisiae protein Erv14, also known as cornichon, belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (S134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14.

Selenoprotein K contributes to CD36 subcellular trafficking in hepatocytes by accelerating nascent COPII vesicle formation and aggravates hepatic steatosis.

SelenoproteinK (SelK), an endoplasmic reticulum (ER) - resident protein, possesses the property of mediate oxidation resistance and ER - associated protein degradation (ERAD) in several tissues. Here, we found that increased SelK markedly promotes fatty acid translocase (CD36) subcellular trafficking and aggravates lipid accumulation in hepatocytes. We demonstrated that SelK is required for the assembly of COPII vesicles and accelerates transport of palmitoylated-CD36 from the ER to Golgi, thus facilitating CD36 plasma membrane distribution both in vivo and in vitro. The mechanism is that SelK increases the stability of Sar1B and triggers CD36-containing nascent COPII vesicle formation, consequently, promotes CD36 subcellular trafficking. Furthermore, we verified that the intervention of SelK SH3 binding domain can inhibit the vesicle formation and CD36 subcellular trafficking, significantly ameliorates NAFLD in mice. Collectively, our findings disclose an unexpected role of SelK in regulating NAFLD development, suggesting that targeting the SelK of hepatocytes may be a new therapeutic strategy for the treatment of NAFLD.

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Loss of TANGO1 Leads to Absence of Bone Mineralization.

TANGO1 (transport and Golgi organization-1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER-Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild-to-moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near-total bone loss and phenocopies the situation of Mia3 -/- mice. Whole-exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4-base pair (4-bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1-related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe-to-lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex.

The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requires SNARE proteins, the homotypic vacuole fusion and protein sorting (HOPS) tethering complex, the RAB7 GTPase Ypt7, and its guanine nucleotide exchange factor (GEF) Mon1-Ccz1. We and others recently identified Ykt6 as the autophagosomal SNARE protein. However, it has not been resolved when and how lipid-anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum (ER)-resident Dsl1 complex and COPII-coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event.

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca2+ and Energy Stresses.

One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca2+ and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca2+ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca2+ mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca2+ stress levels.

How to Avoid a No-Deal ER Exit.

Efficiency and fidelity of protein secretion are achieved thanks to the presence of different steps, located sequentially in time and space along the secretory compartment, controlling protein folding and maturation. After entering into the endoplasmic reticulum (ER), secretory proteins attain their native structure thanks to specific chaperones and enzymes. Only correctly folded molecules are allowed by quality control (QC) mechanisms to leave the ER and proceed to downstream compartments. Proteins that cannot fold properly are instead retained in the ER to be finally destined to proteasomal degradation. Exiting from the ER requires, in most cases, the use of coated vesicles, departing at the ER exit sites, which will fuse with the Golgi compartment, thus releasing their cargoes. Protein accumulation in the ER can be caused by a too stringent QC or by ineffective transport: these situations could be deleterious for the organism, due to the loss of the secreted protein, and to the cell itself, because of abnormal increase of protein concentration in the ER. In both cases, diseases can arise. In this review, we will describe the pathophysiology of protein folding and transport between the ER and the Golgi compartment.

YIPF6 controls sorting of FGF21 into COPII vesicles and promotes obesity.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates glucose, lipid, and energy homeostasis. While gene expression of FGF21 is regulated by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha in the fasted state, little is known about the regulation of trafficking and secretion of FGF21. We show that mice with a mutation in the Yip1 domain family, member 6 gene (Klein-Zschocher [KLZ]; Yipf6KLZ/Y ) on a high-fat diet (HFD) have higher plasma levels of FGF21 than mice that do not carry this mutation (controls) and hepatocytes from Yipf6KLZ/Y mice secrete more FGF21 than hepatocytes from wild-type mice. Consequently, Yipf6KLZ/Y mice are resistant to HFD-induced features of the metabolic syndrome and have increased lipolysis, energy expenditure, and thermogenesis, with an increase in core body temperature. Yipf6KLZ/Y mice with hepatocyte-specific deletion of FGF21 were no longer protected from diet-induced obesity. We show that YIPF6 binds FGF21 in the endoplasmic reticulum to limit its secretion and specifies packaging of FGF21 into coat protein complex II (COPII) vesicles during development of obesity in mice. Levels of YIPF6 protein in human liver correlate with hepatic steatosis and correlate inversely with levels of FGF21 in serum from patients with nonalcoholic fatty liver disease (NAFLD). YIPF6 is therefore a newly identified regulator of FGF21 secretion during development of obesity and could be a target for treatment of obesity and NAFLD.

Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites.

SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet-induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.

COPII vesicles and the expansion of the phagophore.

A new study has identified the proteins that adapt COPII vesicles to the needs of starving cells.

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements.

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4αEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.

O-GlcNAcylation regulates endoplasmic reticulum exit sites through Sec31A modification in conventional secretory pathway.

The conventional secretory pathway is indispensable for eukaryotic cells. Newly synthesized membrane and secretory proteins are released from the endoplasmic reticulum (ER) through ER-derived vesicles to their appropriate destination. Vesicle formation is important for steady protein trafficking. O-GlcNAcylation ( O-GlcNAc) is a unique protein glycosylation signature, whose dynamic regulation by O-GlcNAc transferase and O-GlcNAcase occurs exclusively for nuclear and cytoplasmic proteins. Because of this locally limited property, the role of O-GlcNAc in the conventional protein secretory pathway is unknown. We report that Sec31A on COPII vesicles, a specific coat-protein complex for anterograde trafficking in the ER-Golgi network, is O-GlcNAcylated on S964, which accelerates COPII vesicle formation through control of its binding affinity to apoptosis-linked gene 2, a calcium-binding protein. Together, O-GlcNAc on Sec31A regulates conventional secretory vesicle trafficking in the ER-Golgi network. These modifications accelerate COPII vesicle formation and accelerated anterograde transport of vesicles within the ER-Golgi networks.-Cho, H. J., Mook-Jung, I. O-GlcNAcylation regulates endoplasmic reticulum exit sites through Sec31A modification in conventional secretory pathway.

Retrospective on Cholesterol Homeostasis: The Central Role of Scap.

Scap is a polytopic membrane protein that functions as a molecular machine to control the cholesterol content of membranes in mammalian cells. In the 21 years since our laboratory discovered Scap, we have learned how it binds sterol regulatory element-binding proteins (SREBPs) and transports them from the endoplasmic reticulum (ER) to the Golgi for proteolytic processing. Proteolysis releases the SREBP transcription factor domains, which enter the nucleus to promote cholesterol synthesis and uptake. When cholesterol in ER membranes exceeds a threshold, the sterol binds to Scap, triggering several conformational changes that prevent the Scap-SREBP complex from leaving the ER. As a result, SREBPs are no longer processed, cholesterol synthesis and uptake are repressed, and cholesterol homeostasis is restored. This review focuses on the four domains of Scap that undergo concerted conformational changes in response to cholesterol binding. The data provide a molecular mechanism for the control of lipids in cell membranes.

Autophagosome formation: Where the secretory and autophagy pathways meet.

The upregulation of autophagosome formation in response to nutrient deprivation requires significant intracellular membrane rearrangements that are poorly understood. Recent findings have implicated COPII-coated vesicles, well known as ER-Golgi cargo transport carriers, as key players in macroautophagy. The role of COPII vesicles in macroautophagy and how they interact with autophagy-related (Atg) proteins was unknown. In our recent report, we show that during nutrient deprivation, phosphorylation of the membrane-distal surface of the COPII coat subunit Sec24 facilitates the interaction of Sec24 with the Atg machinery (specifically, Atg9) to regulate the abundance of autophagosomes during starvation. Phosphorylation of Sec24 is specifically required for macroautophagy, but not ER-Golgi transport. These findings begin to unravel the unique function of COPII vesicles during starvation-induced macroautophagy.

Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation.

Endoplasmic Reticulum (ER)-derived COPII coated vesicles constitutively transport secretory cargo to the Golgi. However, during starvation-induced stress, COPII vesicles have been implicated as a membrane source for autophagosomes, distinct organelles that engulf cellular components for degradation by macroautophagy (hereafter called autophagy). How cells regulate core trafficking machinery to fulfill dramatically different cellular roles in response to environmental cues is unknown. Here we show that phosphorylation of conserved amino acids on the membrane-distal surface of the Saccharomyces cerevisiae COPII cargo adaptor, Sec24, reprograms COPII vesicles for autophagy. We also show casein kinase 1 (Hrr25) is a key kinase that phosphorylates this regulatory surface. During autophagy, Sec24 phosphorylation regulates autophagosome number and its interaction with the C-terminus of Atg9, a component of the autophagy machinery required for autophagosome initiation. We propose that the acute need to produce autophagosomes during starvation drives the interaction of Sec24 with Atg9 to increase autophagosome abundance.

Protein quality control at the endoplasmic reticulum.

The ER (endoplasmic reticulum) is the protein folding 'factory' of the secretory pathway. Virtually all proteins destined for the plasma membrane, the extracellular space or other secretory compartments undergo folding and maturation within the ER. The ER hosts a unique PQC (protein quality control) system that allows specialized modifications such as glycosylation and disulfide bond formation essential for the correct folding and function of many secretory proteins. It is also the major checkpoint for misfolded or aggregation-prone proteins that may be toxic to the cell or extracellular environment. A failure of this system, due to aging or other factors, has therefore been implicated in a number of serious human diseases. In this article, we discuss several key features of ER PQC that maintain the health of the cellular secretome.

Intracellular mechanisms of molecular recognition and sorting for transport of large extracellular matrix molecules.

Extracellular matrix (ECM) proteins are biosynthesized in the rough endoplasmic reticulum (rER) and transported via the Golgi apparatus to the extracellular space. The coat protein complex II (COPII) transport vesicles are approximately 60-90 nm in diameter. However, several ECM molecules are much larger, up to several hundreds of nanometers. Therefore, special COPII vesicles are required to coat and transport these molecules. Transmembrane Protein Transport and Golgi Organization 1 (TANGO1) facilitates loading of collagens into special vesicles. The Src homology 3 (SH3) domain of TANGO1 was proposed to recognize collagen molecules, but how the SH3 domain recognizes various types of collagen is not understood. Moreover, how are large noncollagenous ECM molecules transported from the rER to the Golgi? Here we identify heat shock protein (Hsp) 47 as a guide molecule directing collagens to special vesicles by interacting with the SH3 domain of TANGO1. We also consider whether the collagen secretory model applies to other large ECM molecules.