scholarly journals Distinct states of proinsulin misfolding in MIDY

2021 ◽  
Author(s):  
Leena Haataja ◽  
Anoop Arunagiri ◽  
Anis Hassan ◽  
Kaitlin Regan ◽  
Billy Tsai ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Er Quality Control ◽  
Disulfide Bonding ◽  
Interchain Disulfide Bond ◽  
Er Retention ◽  
Allele Specific ◽  
The Many ◽  
Er Export

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.

scholarly journals Distinct states of proinsulin misfolding in MIDY

2021 ◽  
Author(s):  
Leena Haataja ◽  
Anoop Arunagiri ◽  
Anis Hassan ◽  
Kaitlin Regan ◽  
Billy Tsai ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Er Quality Control ◽  
Disulfide Bonding ◽  
Interchain Disulfide Bond ◽  
Er Retention ◽  
Allele Specific ◽  
The Many ◽  
Er Export

AbstractA 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.

Abstract 1070: An Unconventional Vesicular Transport Pathway Regulates the Cell Surface Expression of hERG K + Channels

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Heather A Underkofler ◽  
Sadguna Balijepalli ◽  
Brooke M Moungey ◽  
Jessica K Slind ◽  
Jabe M Best ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Vesicular Transport ◽  
Surface Expression ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Transport Pathway ◽  
Er Export

Approximately 35– 45% of patients that are genotype positive for congenital Long QT Syndrome (LQT) have mutations in the human Ether-a-go-go Related Gene ( hERG ). The purpose of this study was to elucidate the mechanisms that regulate ER export and cell surface expression of hERG channel protein, because these steps are impaired for ~90% of LQT-linked hERG missense mutations. The small GTPases Sar1 and Arf1 regulate the conventional vesicular transport (trafficking) for the ER export of proteins to the Golgi apparatus (Golgi). We generated dominant negative (DN) mutations for Sar1 and Arf1, and co-expressed these DN GTPases with hERG in HEK 293 cells. The trafficking of hERG through the Golgi can be visualized biochemically using Western blot analysis, because additional glycosylation of hERG in the Golgi (Golgi processing) increases the MW of hERG protein from 135kDa to 155kDa. Co-expression of hERG and DN-Sar1 inhibited Golgi processing, decreased hERG current (I hERG ) by 85% compared to control (n≥8 cells per group, p<0.05), and decreased the staining of hERG protein at the cell surface, while co-expression of hERG and DN-Arf1 showed no significant effect on Golgi processing or I hERG . This lack of an effect by DN-Arf1 was selective for hERG as it efficiently blocked the transport of previously reported proteins. Rab11 GTPases regulate the trafficking of proteins from endosomal compartments to the cell surface membrane and/or the Golgi. Rab11a is ubiquitously expressed, whereas Rab11b is expressed primarily in brain and heart. Co-expression of DN-Rab11a did not alter Golgi processing of hERG but reduced I hERG by 51% compared to control (n≥8 cells per group, p<0.05), whereas co-expression of DN-Rab11b inhibited Golgi processing of hERG and reduced I hERG by 79% compared to control (n=8 cells per group, p<0.05). Thus, Rab11a appears to regulate the trafficking of hERG to the cell surface after processing in the Golgi, whereas Rab11b regulates the trafficking of hERG prior to processing in the Golgi. These data suggest that hERG does not traffic via the conventional pathway from the ER to the Golgi, but rather in an unconventional pathway from the ER to endosomal compartments prior to Rab11b-mediated transport to the Golgi and subsequent delivery to the cell membrane.

scholarly journals Inhibiting Endoplasmic Reticulum (ER)-associated Degradation of Misfolded Yor1p Does Not Permit ER Export Despite the Presence of a Diacidic Sorting Signal

2007 ◽  
Vol 18 (9) ◽  
pp. 3398-3413 ◽  
Author(s):  
Silvere Pagant ◽  
Leslie Kung ◽  
Mariana Dorrington ◽  
Marcus C.S. Lee ◽  
Elizabeth A. Miller
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Er Quality Control ◽  
Er Retention ◽  
Copii Vesicles ◽  
Er Export ◽  
Cystic Fibrosis Transmembrane

Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the “B-site” cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-ΔF is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-ΔF by inhibiting its degradation does not permit access of Yor1p-ΔF to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway.

In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1617-1630
Author(s):  
Suk-Won Jin ◽  
Nancy Arno ◽  
Adam Cohen ◽  
Amy Shah ◽  
Qijin Xu ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Rna Binding ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Biochemical Tests ◽  
Cell Fates ◽  
Cytoplasmic Polyadenylation ◽  
Binding Domains ◽  
Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

scholarly journals Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Seung Won Choi ◽  
Yeri Lee ◽  
Kayoung Shin ◽  
Harim Koo ◽  
Donggeon Kim ◽  
...  
Keyword(s):  
Functional Properties ◽  
Therapeutic Target ◽  
Medical Center ◽  
Dominant Negative ◽  
The Cancer Genome Atlas ◽  
Dominant Negative Effect ◽  
Cell Periphery ◽  
Missense Mutations ◽  
Phosphatase Domain ◽  
Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

scholarly journals A disease-associated frameshift mutation in caveolin-1 disrupts caveolae formation and function through introduction of a de novo ER retention signal

2017 ◽  
Vol 28 (22) ◽  
pp. 3095-3111 ◽  
Author(s):  
Courtney A. Copeland ◽  
Bing Han ◽  
Ajit Tiwari ◽  
Eric D. Austin ◽  
James E. Loyd ◽  
...  
Keyword(s):  
Frameshift Mutation ◽  
De Novo ◽  
Dominant Negative ◽  
Caveolin 1 ◽  
Protein Levels ◽  
C Terminus ◽  
Er Retention ◽  
And Function ◽  
Er Retention Signal ◽  
Retention Signal

Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1–/– MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1–/– MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.

scholarly journals Effects of an Interchain Disulfide Bond on Tropomyosin Structure: A Molecular Dynamics Study

2018 ◽  
Vol 19 (11) ◽  
pp. 3376 ◽  
Author(s):  
Natalia A. Koubassova ◽  
Sergey Y. Bershitsky ◽  
Andrey K. Tsaturyan
Keyword(s):  
Heart Failure ◽  
Molecular Dynamics ◽  
Disulfide Bond ◽  
Coiled Coil ◽  
Middle Part ◽  
Actin Binding ◽  
Cross Linking ◽  
Interchain Disulfide Bond ◽  
Structural And Functional Properties ◽  
The Cross

Tropomyosin (Tpm) is a coiled-coil actin-binding dimer protein that participates in the regulation of muscle contraction. Both Tpm chains contain Cys190 residues which are normally in the reduced state, but form an interchain disulfide bond in failing heart. Changes in structural and functional properties of Tpm and its complexes with actin upon disulfide cross-linking were studied using various experimental methods. To understand the molecular mechanism underlying these changes and to reveal the possible mechanism of the involvement of the cross-linking in heart failure, molecular dynamics (MD) simulations of the middle part of Tpm were performed in cross-linked and reduced states. The cross-linking increased bending stiffness of Tpm assessed from MD trajectories at 27 °C in agreement with previous experimental observations. However, at 40 °C, the cross-linking caused a decrease in Tpm stiffness and a significant reduction in the number of main chain hydrogen bonds in the vicinity of residues 133 and 134. These data are in line with observations showing enhanced thermal unfolding of the least stable part of Tpm at 30–40 °C and accelerated trypsin cleavage at residue 133 at 40 °C (but not at 27 °C) upon cross-linking. These results allow us to speculate about the possible mechanism of involvement of Tpm cross-linking to heart failure pathogenesis.

scholarly journals Limited ER quality control for GPI-anchored proteins

2016 ◽  
Vol 213 (6) ◽  
pp. 693-704 ◽  
Author(s):  
Natalia Sikorska ◽  
Leticia Lemus ◽  
Auxiliadora Aguilera-Romero ◽  
Javier Manzano-Lopez ◽  
Howard Riezman ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Misfolded Proteins ◽  
Control Mechanisms ◽  
Gpi Anchor ◽  
Er Quality Control ◽  
Entire Class ◽  
Er Export ◽  
Export Signal

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast. We could efficiently route Gas1* to Hrd1-dependent ERAD and provide evidence that it contains a GPI anchor, ruling out that a GPI anchor obstructs ERAD. Instead, we show that the normally decreased susceptibility of Gas1* to ERAD is caused by canonical remodeling of its GPI anchor, which occurs in all GPI-APs and provides a protein-independent ER export signal. Thus, GPI anchor remodeling is independent of protein folding and leads to efficient ER export of even misfolded species. Our data imply that ER quality control is limited for the entire class of GPI-APs, many of them being clinically relevant.

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