Endoplasmic Reticulum Transmembrane Protein Research Articles

Pro-cathepsin D prevents aberrant protein aggregation dependent on endoplasmic reticulum protein CLN6

We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates.

The role of the ER stress sensor IRE1 in cardiovascular diseases.

Despite enormous advances in the treatment of cardiovascular diseases, including I/R injury and heart failure, heart diseases remain a leading cause of mortality worldwide. Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor endoplasmic reticulum (ER) transmembrane protein that senses ER stress. It manages ER stress induced by the accumulation of unfolded/misfolded proteins via the unfolded protein response (UPR). However, if the stress still persists, the UPR pathways are activated and induce cell death. Emerging evidence shows that, beyond the UPR, IRE1 participates in the progression of cardiovascular diseases by regulating inflammation levels, immunity, and lipid metabolism. Here, we summarize the recent findings and discuss the potential therapeutic effects of IRE1 in the treatment of cardiovascular diseases.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Vernal Keratoconjunctivitis.

Vernal keratoconjunctivitis (VKC) is an ocular allergic disease characterized by a type 2 inflammation, tissue remodeling, and low quality of life for the affected patients. We investigated the involvement of endoplasmic reticulum (ER) stress and unfolded protein response in VKC. Conjunctival imprints from VKC patients and normal subjects (CTs) were collected, and RNA was isolated, reverse transcribed, and analyzed with the Affymetrix microarray. Differentially expressed genes between VKC patients and CTs were evaluated. Genes related to ER stress, apoptosis, and autophagy were further considered. VKC and CT conjunctival biopsies were analyzed by immunohistochemistry (IHC) with specific antibodies against unfolded protein response (UPR), apoptosis, and inflammation. Conjunctival fibroblast and epithelial cell cultures were exposed to the conditioned medium of activated U937 monocytes and analyzed by quantitative PCR for the expression of UPR, apoptosis, autophagy, and inflammatory markers. ER chaperones HSPA5 (GRP78/BiP) and HYOU1 (GRP170) were upregulated in VKC patients compared to CTs. Genes encoding for ER transmembrane proteins, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), ER-associated degradation (ERAD), and autophagy were upregulated, but not those related to apoptosis. Increased positive reactivity of BiP and ATF6 and unchanged expression of apoptosis markers were confirmed by IHC. Cell cultures in stress conditions showed an overexpression of UPR, proinflammatory, apoptosis, and autophagy markers. A significant overexpression of genes encoding for ER stress, UPR, and pro-inflammatory pathway components was reported for VKC. Even though these pathways may lead to ER homeostasis, apoptosis, or inflammation, ER stress in VKC may predominantly contribute to promote inflammation.

Clinical and genetic analysis of trichohepatoneurodevelopmental syndrome caused by a CCDC47 variant

Trichohepatoneurodevelopmental syndrome is an extremely uncommon autosomal recessive disorder resulting from variants in the CCDC47 gene, which encodes a Ca2+-binding endoplasmic reticulum (ER) transmembrane protein. To date, only four patients with CCDC47 deficiency have been reported, all of them with homozygous truncating CCDC47 variants. For this study, a Chinese family was recruited, which included a patient diagnosed with trichohepatoneurodevelopmental syndrome. Whole exome sequencing (WES) identified the proband's novel homozygous CCDC47 variation (NM_020198: c.634C > T(p.Arg212*). The variant was confirmed to be segregating in the proband and her unaffected relatives through Sanger sequencing. The patient described exhibited a clinical phenotype similar to that of patients with the CCDC47 variant. Compared to reported cases with CCDC47 pathogenic variants, our patients showed a novel complication of hearing impairment. In addition, brain abnormalities, small feet, bilateral hip dislocation, hip dysplasia, overlapping toes, pectus excavatum, scoliosis and narrow chest were not observed in our patient. We also examined five different variations and their corresponding phenotypes from five patients, both in current and previous research. Although some clinical manifestations of trichohepatoneurodevelopmental syndrome were highly variable, the most common phenotypes observed in these patients include microcephaly, profound intellectual disability, severe global development delay, pronounced growth restriction, hypotonia, woolly hair, facial dysmorphism, respiratory and visual abnormalities, gastrointestinal abnormalities, liver dysfunction, pruritus, skeletal and limb abnormalities, congenital heart defects and immunodeficiency. The present report is the first of a Chinese infant with homozygous variant in the CCDC47 gene. We expanded the genetic and phenotypic spectrum associated with trichohepatoneurodevelopmental syndrome.

Computer-aided peptide-based drug design for inositol-requiring enzyme 1

Inositol-requiring enzyme 1 (IRE1), an endoplasmic reticulum (ER) transmembrane protein with both kinase and endoribonuclease activities, plays an essential role during ER stress and its subsequent unfolded protein response (UPR). Recent evidence shows IRE1 signaling contributes to tumorigenesis and cancer progression, pointing to the therapeutic importance of this conserved arm of the UPR. Here, we employed different computational tools to design and predict short peptides with the capability of disrupting IRE1 dimerization/oligomerization, as a strategy for inhibiting its Kinase and RNase activities. A mutation-based peptide library was constructed using mCSM-PPI2 and OSPREY 3.0. The molecular interaction analyses between the designed peptides and IRE1 protein were conducted using the HADDOCK 2.2 online server, followed with molecular dynamics analysis by the GROMACS 2020 package. We then selected short peptide candidates that exhibited high affinity and best predicted physicochemical properties in complex with IRE1. Finally, online servers, such as ToxinPred and AllerTop, were used to identify the best peptide candidates that showed no significant allergenic or cytotoxic properties. These rational designed peptides with the capability of binding to IRE1 oligomerization domain can be considered as potential drug candidates for disrupting IRE1 activity in cancer and related diseases, pending for further validation by in silico and experimental studies.

Molekularni mehanizmi uključeni u nastanak stresa endoplazmatskog retikuluma - šta znamo do sada

Endoplasmic reticulum (ER) is an intracellular organelle involved in protein synthesis and folding. When the balance between cell needs for proteins and ER capacity to fold proteins is disrupted, nonfunctional, unfolded, or misfolded proteins accumulate in ER lumen, leading to endoplasmic reticulum stress (ER stress). One of the ways cell uses to overcome ER stress is unfolded protein response (UPR) activation. UPR is initiated by the activation of three ER transmembrane proteins. These proteins are IRE-1a (inositol requiring enzyme-1a), PERK (protein kinase RNA-like endoplasmic reticulum kinase) and ATF6 (activating transcription factor 6) and they are activated when ER chaperone, GRP78 (glucose-regulates protein 78) releases their intraluminal domains. Activation of these transmembrane sensors starts mechanisms that should restore ER function. If ER function is not restored and balance is not achieved, apoptosis is induced in order to maintain cell homeostasis. Activated IRE-1a leads to XBP-1 (X-box binding protein-1) mRNA splicing and activates MAP kinases and inflam-matory pathways that involve nuclear factor cB (NFcB). Activated ATF 6 (ATF6f) functions as a transcriptional factor and increases gene expression for XBP-1, while PERK activation leads to phosphorylation and inactivation of eukaryotic initiation factor 2 (eIF2a) which further leads to decreased protein synthesis. Additionally, eIF2a phosphorylation leads to selective synthesis of ATF4, a transcriptional factor that in irreversibly damaged cells induces cell death activation by C/EBP homologous protein (CHOP) transcription. It is known that ER stress and UPR have a role in different diseases pathogenesis such as diabetes, inflammation, tumors and neurodegenerative diseases. Knowing signaling pathways of UPR and mechanisms by which UPR is involved in diseases pathogenesis can be very significant in targeted therapeutic approaches development.

Progesterone receptor membrane component 1 facilitates Ca2+ signal amplification between endosomes and the endoplasmic reticulum

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.

Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport

Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.

New insights into the effect of VMP1 on the treatment of pressure overload-induced pathological cardiac hypertrophy: Involving SERCA-regulated autophagic flux

Pathological cardiac hypertrophy is an adaptive reaction in response to pressure or volume overload. Autophagy is critical for damage caused by pathological cardiac hypertrophy. Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that is effective in activating autophagy. However, the role of VMP1 in pathological cardiac hypertrophy and its underlying mechanisms remain elusive. This study was designed to explore the potential mechanisms of VMP1 on pressure overload-induced pathological cardiac hypertrophy. In this work, abdominal aorta constriction (AAC) surgery was used to induce pathological cardiac hypertrophy in male C57BL/6 mice. H9C2 cardiomyocytes were treated with phenylephrine stimulation (PE) to induce the hypertrophic response. The in vivo results revealed that mice with AAC surgery caused pathological cardiac hypertrophy as evidenced by improved cardiac function according to multiple echocardiographic parameters. Moreover, elevated VMP1 expression was also observed in mice after AAC surgery. VMP1 knockdown aggravated changes in cardiac structure, cardiac dysfunction, and fibrosis. Meanwhile, VMP1 knockdown suppressed autophagy and endoplasmic reticulum calcium ATPase (SERCA) activity in heart tissues. H9C2 cardiomyocytes with VMP1 overexpression were used to investigate the specific mechanism of VMP1 in pathological cardiac hypertrophy, and VMP1 overexpression increased autophagic flux by upregulating SERCA activity. In conclusion, these findings revealed that VMP1 protected against pressure overload-induced pathological cardiac hypertrophy by inducing SERCA-regulated autophagic flux. Our results provide valuable insights regarding the pathophysiology of pathological cardiac hypertrophy and clues to a novel target for the treatment of pathological cardiac hypertrophy.

TMEM41B Is an Interferon-Stimulated Gene That Promotes Pseudorabies Virus Replication.

Pseudorabies virus (PRV) is a double-stranded DNA virus that causes Aujeszky's disease and is responsible for economic loss worldwide. Transmembrane protein 41B (TMEM41B) is a novel endoplasmic reticulum (ER)-localized regulator of autophagosome biogenesis and lipid mobilization; however, the role of TMEM41B in regulating PRV replication remains undocumented. In this study, PRV infection was found to upregulate TMEM41B mRNA and protein levels both in vitro and in vivo. For the first time, we found that TMEM41B could be induced by interferon (IFN), suggesting that TMEM41B is an IFN-stimulated gene (ISG). While TMEM41B knockdown suppressed PRV proliferation, TMEM41B overexpression promoted PRV proliferation. We next studied the specific stages of the virus life cycle and found that TMEM41B knockdown affected PRV entry. Mechanistically, we demonstrated that the knockdown of TMEM41B blocked PRV-stimulated expression of the key enzymes involved in lipid synthesis. Additionally, TMEM41B knockdown played a role in the dynamics of lipid-regulated PRV entry-dependent clathrin-coated pits (CCPs). Lipid replenishment restored the CCP dynamic and PRV entry in TMEM41B knockdown cells. Together, our results indicate that TMEM41B plays a role in PRV infection via regulating lipid homeostasis. IMPORTANCE PRV belongs to the alphaherpesvirus subfamily and can establish and maintain a lifelong latent infection in pigs. As such, an intermittent active cycle presents great challenges to the prevention and control of PRV disease and is responsible for serious economic losses to the pig breeding industry. Studies have shown that lipids play a crucial role in PRV proliferation. Thus, the manipulation of lipid metabolism may represent a new perspective for the prevention and treatment of PRV. In this study, we report that the ER transmembrane protein TMEM41B is a novel ISG involved in PRV infection by regulating lipid synthesis. Therefore, our findings indicate that targeting TMEM41B may be a promising approach for the development of PRV vaccines and therapeutics.

Abstract ND09: M3913 induces the maladaptive unfolded protein response through a novel mechanism resulting in strong anti-tumor activity

Abstract Therapeutic strategies based on exacerbated stress signaling may represent novel and effective treatment paradigms in oncology. Here, we disclose for the first time the endoplasmic reticulum (ER) stress modulator (ERSM) M3913 that was discovered and developed following a phenotypic screening campaign. Mechanistically, M3913 engages an ER transmembrane protein not yet implicated in cancer biology to induce a Ca2+ shift from the ER towards the cytoplasm, resulting in an unfolded protein response and subsequent antitumor activity in sensitive preclinical models. We detected concurrent elevation of bona fide ER stress markers by several methods including qRT-PCR, Western Blotting analysis, and unbiased transcriptome analysis. Genetic removal of M3913’s putative ER transmembrane target abrogated the M3913-mediated, but not the Thapsigargin-mediated, ER stress response. PK/PD studies confirmed a dose- and time-dependent upregulation of ER stress markers in preclinical cancer models in vivo. As a monotherapy, M3913 induced full and partial tumor regression in preclinical models of multiple myeloma, non-small-cell lung cancer, triple-negative breast cancer, and other cancers. An in vitro combination screen indicated combination potential of M3913 with standard-of-care agents and novel candidate drugs. IND-enabling toxicology and additional mechanism-of-action studies in rat and minipig uncovered specific target organs for M3913 in line with expression of the target in human tissues. This presentation details mechanistic profiling data from M3913, reveals its putative ER-resident target and discusses forward translation studies that support M3913 as a novel therapeutic option for hard-to-treat subtypes of a variety of cancers. Citation Format: Frank Czauderna, Richard Schneider, Shivapriya Ramaswamy, Susanne Brandstetter, Elise Drouin, Olga Bogatyrova, Johanna Mazur, Qing Sun, Catherine Jorand-Lebrun, Eva Sherbetjian, Jonny Nachtigall, Carolyn Wing, Christian Hildebrand, Peter Ellinghaus, Isabelle Lemoine, Mac Johnson, Russell Hoover, Michael Clark, Ralph Lindemann. M3913 induces the maladaptive unfolded protein response through a novel mechanism resulting in strong anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND09.

PDZD8-mediated endoplasmic reticulum–mitochondria associations regulate sympathetic drive and blood pressure through the intervention of neuronal mitochondrial homeostasis in stress-induced hypertension

Neuronal hyperexcitation in the rostral ventrolateral medulla (RVLM) drives heightened sympathetic nerve activity and contributes to the etiology of stress-induced hypertension (SIH). Maintenance of mitochondrial functions is central to neuronal homeostasis. PDZD8, an endoplasmic reticulum (ER) transmembrane protein, tethers ER to mitochondria. However, the mechanisms of PDZD8-mediated ER–mitochondria associations regulating neuronal mitochondrial functions and thereby mediating blood pressure (BP) in the RVLM of SIH were largely unknown. SIH rats were subjected to intermittent electric foot shocks plus noise for 2 h twice daily for 15 consecutive days. The underlying mechanisms of PDZD8 were investigated through in vitro experiments by using small interfering RNA and through in vivo experiments, such as intra-RVLM microinjection and Western blot analysis. The function of PDZD8 on BP regulation in the RVLM was determined in vivo via the intra-RVLM microinjection of adeno-associated virus (AAV)2-r-Pdzd8. We found that the c-Fos-positive RVLM tyrosine hydroxylase (TH) neurons, renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE) level, BP, and heart rate (HR) were elevated in SIH rats. ER–mitochondria associations in RVLM neurons were significantly reduced in SIH rats. PDZD8 was mainly expressed in RVLM neurons, and mRNA and protein levels were markedly decreased in SIH rats. In N2a cells, PDZD8 knockdown disrupted ER–mitochondria associations and mitochondrial structure, decreased mitochondrial membrane potential (MMP) and respiratory metabolism, enhanced ROS levels, and reduced catalase (CAT) activity. These effects suggested that PDZD8 dysregulation induced mitochondrial malfunction. By contrast, PDZD8 upregulation in the RVLM of SIH rats could rescue neuronal mitochondrial function, thereby suppressing c-Fos expression in TH neurons and decreasing RSNA, plasma NE, BP, and HR. Our results indicated that the dysregulation of PDZD8-mediated ER–mitochondria associations led to the loss of the activity homeostasis of RVLM neurons by disrupting mitochondrial functions, thereby participating in the regulation of SIH pathology.

VMP1 prevents Ca2+ overload in endoplasmic reticulum and maintains naive T cell survival.

Ca2+ in endoplasmic reticulum (ER) dictates T cell activation, proliferation, and function via store-operated Ca2+ entry. How naive T cells maintain an appropriate level of Ca2+ in ER remains poorly understood. Here, we show that the ER transmembrane protein VMP1 is essential for maintaining ER Ca2+ homeostasis in naive T cells. VMP1 promotes Ca2+ release from ER under steady state, and its deficiency leads to ER Ca2+ overload, ER stress, and secondary Ca2+ overload in mitochondria, resulting in massive apoptosis of naive T cells and defective T cell response. Aspartic acid 272 (D272) of VMP1 is critical for its ER Ca2+ releasing activity, and a knockin mouse strain with D272 mutated to asparagine (D272N) demonstrates all functions of VMP1 in T cells in vivo depend on its regulation of ER Ca2+. These data uncover an indispensable role of VMP1 in preventing ER Ca2+ overload and maintaining naive T cell survival.

The Effect of BSCL2 Gene on Fat Deposition Traits in Pigs.

BSCL2 encodes seipin, a transmembrane endoplasmic reticulum protein associated with lipodystrophy and severe metabolic complications, including diabetes and hepatic steatosis. In pigs, BSCL2 expression increases during adipocyte differentiation. In the present study, we identified significant gene variants associated with fat deposition (FD)-related processes based on subcutaneous fat tissue RNA-seq data. In the association study, to prove our hypothesis, three Polish pig breeds were included: Złotnicka White (ZW, n = 72), Polish Landrace (PL, n = 201), and Polish Large White (PLW, n = 169). Based on variant calling analysis and χ2 tests, BSCL2 mutations showing significantly different genotype/allele distribution between high- and low-fat pigs were selected for a comprehensive association study. Four interesting BSCL2 variants (rs346079334, rs341493267, rs330154033, and rs81333153) belonging to downstream and missense mutations were investigated. Our study showed a significant decrease in minor allele frequency for two BSCL2 variants (rs346079334 and rs341493267) in PL pigs in 2020-2021. In ZW, BSCL2 mutations significantly affected loin and ham fats, meat redness, and growth performance traits, such as feed conversion and daily feed intake. Similar observations were noted for PLW and PL, where BSCL2 mutations influenced fat depositions and meat traits, such as loin eye area, loin mass and fat, carcass yield, and growth performance traits. Based on the observation in pigs, our study supports the theory that BSCL2 expressed in subcutaneous fat is involved in the FD process.

S-acylation of SARAF regulates store-operated calcium entry.

The endoplasmic reticulum (ER) is the primary intracellular calcium storage site. Upon cellular stimulation by an agonist, the production of inositol 1,4,5-trisphosphate (IP3) leads to activation of IP3 receptor channels on the ER membrane and calcium efflux from ER lumen. This leads to ER calcium store depletion. Upon store depletion, the ER-resident calcium sensor STIM1 and plasma membrane calcium channel Orai1 bind to each other to form the calcium release-activated calcium (CRAC) channel. These channels lead to calcium entry from extracellular milieu. This entry is known as store-operated calcium entry (SOCE). Previously, we have shown that Orai1 and STIM1 are S-acylated upon store-depletion and this process is crucial for CRAC channel formation and SOCE. TMEM66, also known as SARAF, is a ER transmembrane protein that is known to associate with STIM1 in resting conditions, and subsequently with Orai1 after store-depletion. This binding of SARAF with Orai1 upon store-depletion is important for the inactivation of CRAC currents. We hypothesize S-acylation of SARAF is required for the inactivation of SOCE. Here we show that SARAF is S-acylated in Jurkat T cells upon activation of the T cell receptor (TCR) and subsequent store-depletion. We also show the residue where SARAF undergoes this S-acylation. Importantly, a C320S mutant version of SARAF that cannot undergo S-acylation has deficits in CRAC channel formation and SOCE. Put together, these data suggest SARAF is a crucial component of SOCE.

ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis, leading to impairment of endoplasmic reticulum (ER) function and activating the unfolded protein response (UPR). PARP16 is an active (ADP-ribosyl)transferase known tail-anchored ER transmembrane protein with a cytosolic catalytic domain. Here, we find PARP16 is highly expressed in ischemic cerebral hemisphere and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated immortalized hippocampal neuronal cell HT22. Using an adeno-associated virus-mediated PARP16 knockdown approach in mice, we find PARP16 knockdown decreases infarct demarcations and has a better neurological outcome after ischemic stroke. Our data indicate PARP16 knockdown decreases ER stress and neuronal death caused by OGD/R, whereas PARP16 overexpression promotes ER stress-mediated cell damage in primary cortical neurons. Furthermore, PARP16 functions mechanistically as ADP-ribosyltransferase to modulate the level of ADP-ribosylation of the corresponding PERK and IRE1α arm of the UPR, and such modifications mediate activation of PERK and IRE1α. Indeed, pharmacological stimulation of the UPR using Brefeldin A partly counteracts PARP16 knockdown-mediated neuronal protection upon OGD/R treatment. In conclusion, PARP16 plays a crucial role in post-ischemic UPR and PARP16 knockdown alleviates brain injury after ischemic stroke. This study demonstrates the potential of the PARP16-PERK/IRE1α axis as a target for neuronal survival in ischemic stroke.

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.

PARP16-Mediated Stabilization of Amyloid Precursor Protein mRNA Exacerbates Alzheimer's Disease Pathogenesis.

The accumulation and deposition of beta-amyloid (Aβ) are key neuropathological hallmarks of Alzheimer's disease (AD). PARP16, a Poly(ADP-ribose) polymerase, is a known tail-anchored endoplasmic reticulum (ER) transmembrane protein that transduces ER stress during pathological processes. Here, we found that PARP16 was significantly increased in the hippocampi and cortices of APPswe/PS1dE9 (APP/PS1) mice and hippocampal neuronal HT22 cells exposed to Aβ, suggesting a positive correlation between the progression of AD pathology and the overexpression of PARP16. To define the effect of PARP16 on AD progression, adeno-associated virus mediated-PARP16 knockdown was used in APP/PS1 mice to investigate the role of PARP16 in spatial memory, amyloid burden, and neuroinflammation. Knockdown of PARP16 partly attenuated impaired spatial memory, as indicated by the Morris water maze test, and decreased amyloid deposition, neuronal apoptosis, and the production of inflammatory cytokines in the brains of APP/PS1 mice. In vitro experiments demonstrated that the knockdown of PARP16 expression rescued neuronal damage and ER stress triggered by Aβ. Furthermore, we discovered that intracellular PARP16 acts as an RNA-binding protein that regulates the mRNA stability of amyloid precursor protein (APP) and protects targeted APP from degradation, thereby increasing APP levels and AD pathology. Our findings revealed an unanticipated role of PARP16 in the pathogenesis of AD, and at least in part, its association with increased APP mRNA stability.

PERK/ATF3-Reduced ER Stress on high potassium environment in the suppression of tumor ferroptosis.

Potassium (K+) is a vital intracellular cation. In the human body, it regulates membrane potential, electrical excitation, protein synthesis, and cell death. Recent studies revealed that dying cancer cells release potassium into the tumor microenvironment (TME), thereby influencing cell survival-related events. Several investigations reported that potassium channels and high potassium levels influence apoptosis. Increasing extracellular potassium and inhibiting K+ efflux channels significantly block the apoptotic machinery. However, it is unknown whether a high-potassium environment also affects other types of cell death such as ferroptosis. In the present study, cell counting kit (CCK-8), colony formation ability, and 5-ethynyl-2'-deoxyuridine (EdU) assays demonstrated that a high-potassium environment reverses erastin-induced ferroptosis. RNA sequencing (RNA-Seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses indicated that high potassium levels attenuated the unfolded protein response that is characteristic of endoplasmic reticulum (ER) stress. The ER transmembrane proteins PRKR-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) are recognized as ER stress sensors. Here, the PERK blocker GSK2606414 significantly rescued ferroptosis. The present work also disclosed that the ER-related gene activating transcription factor 3 (ATF3) played a vital role in regulating ferroptosis in a high-potassium environment. The foregoing results revealed the roles of potassium and the TME in cancer cell ferroptosis and provided a potential clinical therapeutic strategy for cancer.

Intercellular transfer of activated STING triggered by RAB22A-mediated non-canonical autophagy promotes antitumor immunity

STING, an endoplasmic reticulum (ER) transmembrane protein, mediates innate immune activation upon cGAMP stimulation and is degraded through autophagy. Here, we report that activated STING could be transferred between cells to promote antitumor immunity, a process triggered by RAB22A-mediated non-canonical autophagy. Mechanistically, RAB22A engages PI4K2A to generate PI4P that recruits the Atg12–Atg5–Atg16L1 complex, inducing the formation of ER-derived RAB22A-mediated non-canonical autophagosome, in which STING activated by agonists or chemoradiotherapy is packaged. This RAB22A-induced autophagosome fuses with RAB22A-positive early endosome, generating a new organelle that we name Rafeesome (RAB22A-mediated non-canonical autophagosome fused with early endosome). Meanwhile, RAB22A inactivates RAB7 to suppress the fusion of Rafeesome with lysosome, thereby enabling the secretion of the inner vesicle of the autophagosome bearing activated STING as a new type of extracellular vesicle that we define as R-EV (RAB22A-induced extracellular vesicle). Activated STING-containing R-EVs induce IFNβ release from recipient cells to the tumor microenvironment, promoting antitumor immunity. Consistently, RAB22A enhances the antitumor effect of the STING agonist diABZI in mice, and a high RAB22A level predicts good survival in nasopharyngeal cancer patients treated with chemoradiotherapy. Our findings reveal that Rafeesome regulates the intercellular transfer of activated STING to trigger and spread antitumor immunity, and that the inner vesicle of non-canonical autophagosome originated from ER is secreted as R-EV, providing a new perspective for understanding the intercellular communication of organelle membrane proteins.