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Item Distinct states of proinsulin misfolding in MIDY(Springer, 2021-08) Haataja, Leena; Arunagiri, Anoop; Hassan, Anis; Regan, Kaitlin; Tsai, Billy; Dhayalan, Balamurugan; Weiss, Michael A.; Liu, Ming; Arvan, Peter; Biochemistry and Molecular Biology, School of MedicineA precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.Item Enhanced Ca2+-channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease(Springer Nature, 2023-03-27) Thoudam, Themis; Chanda, Dipanjan; Lee, Jung Yi; Jung, Min-Kyo; Sinam, Ibotombi Singh; Kim, Byung-Gyu; Park, Bo-Yoon; Kwon, Woong Hee; Kim, Hyo-Jeong; Kim, Myeongjin; Lim, Chae Won; Lee, Hoyul; Huh, Yang Hoon; Miller, Caroline A.; Saxena, Romil; Skill, Nicholas J.; Huda, Nazmul; Kusumanchi, Praveen; Ma, Jing; Yang, Zhihong; Kim, Min-Ji; Mun, Ji Young; Harris, Robert A.; Jeon, Jae-Han; Liangpunsakul, Suthat; Lee, In-Kyu; Pathology and Laboratory Medicine, School of MedicineCa2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in hepatic GRP75-mediated mitochondria-associated ER membrane (MAM) Ca2+-channeling (MCC) complex formation promotes mitochondrial dysfunction in vitro and in male mouse model of ALD. Unbiased transcriptomic analysis reveals PDK4 as a prominently inducible MAM kinase in ALD. Analysis of human ALD cohorts further corroborate these findings. Additional mass spectrometry analysis unveils GRP75 as a downstream phosphorylation target of PDK4. Conversely, non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced MCC complex formation and subsequent mitochondrial Ca2+ accumulation and dysfunction. Finally, ectopic induction of MAM formation reverses the protective effect of PDK4 deficiency in alcohol-induced liver injury. Together, our study defines a mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.Item Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones(Public Library of Science, 2023-04-17) Koppes, Erik A.; Johnson, Marie A.; Moresco, James J.; Luppi, Patrizia; Lewis, Dale W.; Stolz, Donna B.; Diedrich, Jolene K.; Yates, John R., III; Wek, Ronald C.; Watkins, Simon C.; Gollin, Susanne M.; Park, Hyun Jung; Drain, Peter; Nicholls, Robert D.; Biochemistry and Molecular Biology, School of MedicinePrader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in β-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS β-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS β-cells. Consistent with reduced ER chaperones levels, PWS INS-1 β-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS β-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic β-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and β-cell secretory pathway function.Item Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L(Wiley, 2017-01) Justis, Anna V.; Hansen, Bryan; Beare, Paul A.; King, Kourtney B.; Heinzen, Robert A.; Gilk, Stacey D.; Microbiology and Immunology, School of MedicineCoxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol-binding protein-related protein 1 long (ORP1L) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.Item Recruitment and function of ORP1L on the Coxiella burnetii parasitophorous vacuole(2017-12-07) Justis, Anna Victoria; Gilk, Stacey D.; Spinola, Stanley M.; Nelson, David; Arrizabalaga, Gustavo A.; Harrington, Maureen A.Coxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.Item Regulation of endoplasmic reticulum calcium homeostasis in pancreatic β cells(2016-06-21) Tong, Xin; Evans-Molina, Carmella; Day, Richard; Tune, Johnathan; Fueger, Patrick T.; Dong, X. CharlieDiabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity.Item Role of ATF4 in directing gene expression in the basal state and during the unfolded protein response in liver(2016-08) Fusakio, Michael Edward; Wek, Ronald C.Disturbances in membrane composition and protein folding in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR). Three UPR sensory proteins, PERK (PEK/EIF2AK3), IRE1, and ATF6 are each activated by ER stress. PERK phosphorylation of the alpha subunit of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with the preferential translation of ATF4 (CREB2). Results from cultured cells demonstrate that ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterized two ATF4 knockout mouse models and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but rather ATF6 is a primary inducer. RNA-sequence analysis indicated that ATF4 was responsible for a small portion of the PERK-dependent genes in the UPR. This smaller than expected subset of gene expression lends itself to the relevance of UPR crosstalk, with ATF6, XBP1, and CHOP being capable of upregulating UPR genes in the absence of ATF4. RNA-sequence analysis also revealed a requirement for expression of ATF4 for expression of a comparable number of genes basally, including those involved in oxidative stress response and cholesterol metabolism. Consistent with this pattern of gene expression, loss of ATF4 in our mouse model resulted in enhanced oxidative damage and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera. Taken together, this study highlights both an expansion of the role of ATF4 in transcriptional regulation of genes involved in metabolism in the basal state and a more specialized role during ER stress. These findings are important for understanding the variances of the UPR signaling between cell culture and in vivo and for a greater understanding of all the roles ATF4 plays within the cell.Item SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells(Springer, 2023) Iida, Hitoshi; Kono, Tatsuyoshi; Lee, Chih‑Chun; Krishnan, Preethi; Arvin, Matthew C.; Weaver, Staci A.; Jarvela, Timothy S.; Branco, Renato C. S.; McLaughlin, Madeline R.; Bone, Robert N.; Tong, Xin; Arvan, Peter; Lindberg, Iris; Evans‑Molina, Carmella; Medicine, School of MedicineAims/hypothesis: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. Methods: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. Results: βS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. Conclusions/interpretation: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. Data availability: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).Item The Effect of SERCA Activation on Functional Characteristics and Signaling of Rat Soleus Muscle upon 7 Days of Unloading(MDPI, 2023-09-06) Sharlo, Kristina A.; Lvova, Irina D.; Tyganov, Sergey A.; Zaripova, Ksenia A.; Belova, Svetlana P.; Kostrominova, Tatiana Y.; Shenkman, Boris S.; Nemirovskaya, Tatiana L.; Anatomy, Cell Biology and Physiology, School of MedicineSkeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.Item The unfolded protein response regulates hepatocellular injury during the pathogenesis of nonalcoholic steatohepatitis(2016-08) Willy, Jeffrey Allen; Wek, Ronald C.Non-alcoholic steatohepatitis (NASH), which is characterized by the induction of hepatocellular death and inflammation, is associated with the activation of cellular stress pathways such as the Unfolded Protein Response (UPR), an adaptive response to disruptions in endoplasmic reticulum (ER) homeostasis. Because the role of the UPR in the progression of liver disease is not well understood, we established an in vitro model to evaluate the role of the UPR in NASH and translated results to clarify disease progression in human liver biopsy samples. Treating HepG2 cells and primary human hepatocytes with saturated, but not unsaturated free fatty acids (FFAs), at physiologic concentrations induced hepatotoxicity by inhibiting autophagic flux. Saturated FFA treatment activated the UPR, including the transcription factors CHOP (GADD153/DDIT3) and NF-κB, leading to increased expression and secretion of cytokines such as TNFα and IL-8 that contributed to hepatic cell death and inflammation. Depletion of either CHOP or the RELA subunit of NF-κB in hepatocytes alleviated autophagy and cytokine secretion, resulting in enhanced cell viability and lowered inflammatory responses during exposure to saturated FFAs. We carried out next generation sequencing on cells deleted for either CHOP or RELA and identified IBTKα as a novel UPR member directly regulated by CHOP and NF-κB. In response to saturated FFAs, loss of IBTKα increased cell survival through lowered phagophore formation and reduced cytokine secretion. We also identified binding partners of IBTKα by immunoprecipitation and LC/MS, indicating that that IBTKα is part of a protein complex which functions at ER exit sites to facilitate initiation of autophagy and protein secretion. Furthermore, we discovered that CHOP and RELA coordinately regulate proteasome activity through NRF2 as an adaptive response to an inhibition of autophagic flux following palmitate exposure. To validate our model, we utilized human liver biopsy samples and demonstrated up-regulation of the UPR coincident with accumulation of autophagy markers, as well as secretion of cytokines IL 8 and TNFα in serum of NASH patients. Our study provides a mechanistic understanding of the roles of the UPR and autophagy in regulating saturated FFA induced hepatotoxicity at the cellular level.