Browsing by Subject "Endoplasmic reticulum"

Now showing 1 - 10 of 19
  • Thumbnail Image
    Item
    Advanced Imaging Techniques for the Characterization of Subcellular Organelle Structure in Pancreatic Islet β Cells
    (Wiley, 2023-12-29) McLaughlin, Madeline R.; Weaver, Staci A.; Syed, Farooq; Evans-Molina, Carmella; Pediatrics, School of Medicine
    Type 2 diabetes (T2D) affects more than 32.3 million individuals in the United States, creating an economic burden of nearly $966 billion in 2021. T2D results from a combination of insulin resistance and inadequate insulin secretion from the pancreatic β cell. However, genetic and physiologic data indicate that defects in β cell function are the chief determinant of whether an individual with insulin resistance will progress to a diagnosis of T2D. The subcellular organelles of the insulin secretory pathway, including the endoplasmic reticulum, Golgi apparatus, and secretory granules, play a critical role in maintaining the heavy biosynthetic burden of insulin production, processing, and secretion. In addition, the mitochondria enable the process of insulin release by integrating the metabolism of nutrients into energy output. Advanced imaging techniques are needed to determine how changes in the structure and composition of these organelles contribute to the loss of insulin secretory capacity in the β cell during T2D. Several microscopy techniques, including electron microscopy, fluorescence microscopy, and soft X-ray tomography, have been utilized to investigate the structure-function relationship within the β cell. In this overview article, we will detail the methodology, strengths, and weaknesses of each approach.
  • Thumbnail Image
    Item
    Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis
    (Public Library of Science, 2012) Chen, Xi; Li, Min; Chen, Daohong; Gao, Wentao; Guan, Jun-Lin; Komatsu, Massaki; Yin, Xiao-Ming; Pathology and Laboratory Medicine, School of Medicine
    Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.
  • Thumbnail Image
    Item
    Coordinated signaling of activating transcription factor 6α and inositol-requiring enzyme 1α regulates hepatic stellate cell-mediated fibrogenesis in mice
    (American Physiological Society, 2021) Xue, Fei; Lu, Jianwen; Buchl, Samuel C.; Sun, Liankang; Shah, Vijay H.; Malhi, Harmeet; Maiers, Jessica L.; Medicine, School of Medicine
    Liver injury and the unfolded protein response (UPR) are tightly linked, but their relationship differs with cell type and injurious stimuli. UPR initiation promotes hepatic stellate cell (HSC) activation and fibrogenesis, but the underlying mechanisms are unclear. Despite the complexity and overlap downstream of UPR transducers inositol-requiring protein 1α (IRE1α), activating transcription factor 6α (ATF6α), and protein kinase RNA-like ER kinase (PERK), previous research in HSCs primarily focused on IRE1α. Here, we investigated the fibrogenic role of ATF6α or PERK in vitro and HSC-specific UPR signaling in vivo. Overexpression of ATF6α, but not the PERK effector activating transcription factor 4 (ATF4), promoted HSC activation and fibrogenic gene transcription in immortalized HSCs. Furthermore, ATF6α inhibition through Ceapin-A7, or Atf6a deletion, disrupted transforming growth factor β (TGFβ)-mediated activation of primary human hepatic stellate cells (hHSCs) or murine hepatic stellate cells (mHSCs), respectively. We investigated the fibrogenic role of ATF6α in vivo through conditional HSC-specific Atf6a deletion. Atf6aHSCΔ/Δ mice displayed reduced fibrosis and HSC activation following bile duct ligation (BDL) or carbon tetrachloride (CCl4)-induced injury. The Atf6aHSCΔ/Δ phenotype differed from HSC-specific Ire1a deletion, as Ire1aHSCΔ/Δ mice showed reduced fibrogenic gene transcription but no changes in fibrosis compared with Ire1afl/fl mice following BDL. Interestingly, ATF6α signaling increased in Ire1aΔ/Δ HSCs, whereas IRE1α signaling was upregulated in Atf6aΔ/Δ HSCs. Finally, we asked whether co-deletion of Atf6a and Ire1a additively limits fibrosis. Unexpectedly, fibrosis worsened in Atf6aHSCΔ/ΔIre1aHSCΔ/Δ mice following BDL, and Atf6aΔ/ΔIre1aΔ/Δ mHSCs showed increased fibrogenic gene transcription. ATF6α and IRE1α individually promote fibrogenic transcription in HSCs, and ATF6α drives fibrogenesis in vivo. Unexpectedly, disruption of both pathways sensitizes the liver to fibrogenesis, suggesting that fine-tuned UPR signaling is critical for regulating HSC activation and fibrogenesis. NEW & NOTEWORTHY: ATF6α is a critical driver of hepatic stellate cell (HSC) activation in vitro. HSC-specific deletion of Atf6a limits fibrogenesis in vivo despite increased IRE1α signaling. Conditional deletion of Ire1α from HSCs limits fibrogenic gene transcription without impacting overall fibrosis. This could be due in part to observed upregulation of the ATF6α pathway. Dual loss of Atf6a and Ire1a from HSCs worsens fibrosis in vivo through enhanced HSC activation.
  • Thumbnail Image
    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 Medicine
    A 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.
  • Thumbnail Image
    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 Medicine
    Ca2+ 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.
  • Thumbnail Image
    Item
    Host-targeting of virulence determinants and phosphoinositides in blood stage malaria parasites
    (Elsevier, 2012) Bhattacharjee, Souvik; Stahelin, Robert V.; Haldar, Kasturi; Biochemistry and Molecular Biology, School of Medicine
    Blood stage malaria parasites target a 'secretome' of hundreds of proteins including virulence determinants containing a host (cell) targeting (HT) signal, to human erythrocytes. Recent studies reveal that the export mechanism is due to the HT signal binding to the lipid phosphatidylinositol-3-phosphate [PI(3)P] in the parasite endoplasmic reticulum (ER). An aspartic protease plasmepsin V which cleaves a specialized form of the HT signal was previously thought to be the export mechanism, but is now recognized as a dedicated peptidase that cleaves the signal anchor subsequent to PI(3)P binding. We discuss a model of PI(3)P-dependent targeting and PI(3)P biology of a major human pathogen.
  • Thumbnail Image
    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 Medicine
    Prader-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.
  • Thumbnail Image
    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 Medicine
    Coxiella 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.
  • Thumbnail Image
    Item
    Islet β-Cell Endoplasmic Reticulum Stress Precedes the Onset of Type 1 Diabetes in the Nonobese Diabetic Mouse Model
    (American Diabetes Association, 2012) Tersey, Sarah A.; Nishiki, Yurika; Templin, Andrew T.; Cabrera, Susanne M.; Stull, Natalie D.; Colvin, Stephanie C.; Evans-Molina, Carmella; Rickus, Jenna L.; Maier, Bernhard; Mirmira, Raghavendra G.; Pediatrics, School of Medicine
    Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.
  • Thumbnail Image
    Item
    Mitochondria and Endoplasmic Reticulum in Diabetes and Its Complications
    (Hindawi, 2012) Lee, Ki-Up; Harris, Robert A.; Biochemistry and Molecular Biology, School of Medicine