scholarly journals The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

2007 ◽  
Vol 401 (3) ◽  
pp. 701-709 ◽  
Author(s):  
Matthew P. A. Henderson ◽  
Yeen Ting Hwang ◽  
John M. Dyer ◽  
Robert T. Mullen ◽  
David W. Andrews
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Lipid Bilayers ◽  
Fusion Proteins ◽  
Molecular Mechanisms ◽  
Cytochrome B5 ◽  
Terminal Sequence ◽  
C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

scholarly journals Dehydroepiandrosterone Effect on Toxoplasma gondii: Molecular Mechanisms Associated to Parasite Death

2021 ◽  
Author(s):  
Jorge Morales-Montor
Keyword(s):  
Toxoplasma Gondii ◽  
Molecular Mechanisms ◽  
Cytochrome B5 ◽  
Protozoan Parasite ◽  
Intracellular Parasites ◽  
Sds Page ◽  
Parasitic Load ◽  
Dhea Treatment

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasite Toxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals. Dehydroepiandrosterone (DHEA) has proved to be an important molecule that could drive resistance against a variety of infections, including intracellular parasites such as Plasmodium falciparum and Trypanozoma cruzi, among others. However, to date it has not been explored the role of DHEA on T. gondii. In here, we demonstrated for the first time the toxoplasmicidal effect of DHEA on extracellular tachyzoites. Ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading to the loss of the organelle structure and organization, as well as the loss of the cellular shape. In vitro treatment with DHEA reduces the viability of extracellular tachyzoites and passive invasion process. 2D SDS-PAGE analysis revealed that in the presence of the hormone a progesterone receptor membrane component (PGRMC) with a cytochrome b5 family heme/steroid binding domain-containing protein was expressed, while the expression of proteins that are essential for motility and virulence was highly reduced. Finally, in vivo DHEA treatment induced a reduction of parasitic load in male, but not in female mice.

scholarly journals CaMKII as a pathological mediator of ER stress, oxidative stress, and mitochondrial dysfunction in a murine model of nephronophthisis

2016 ◽  
Vol 310 (11) ◽  
pp. F1414-F1422 ◽  
Author(s):  
Christina Bracken ◽  
Philippe Beauverger ◽  
Olivier Duclos ◽  
Ryan J. Russo ◽  
Kelly A. Rogers ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Oxidative Damage ◽  
Molecular Mechanisms ◽  
Kidney Diseases ◽  
Cystic Kidney ◽  
Effective Inhibition ◽  
Stress Oxidative

Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.

scholarly journals Dehydroepiandrosterone Effect on Toxoplasma gondii: Molecular Mechanisms Associated to Parasite Death

2021 ◽  
Vol 9 (3) ◽  
pp. 513
Author(s):  
Saé Muñiz-Hernández ◽  
Angélica Luna-Nophal ◽  
Carmen T. Gómez-De León ◽  
Lenin Domínguez-Ramírez ◽  
Olga A. Patrón-Soberano ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Molecular Mechanisms ◽  
Cytochrome B5 ◽  
Protozoan Parasite ◽  
Intracellular Parasites ◽  
Sds Page ◽  
Parasitic Load ◽  
Dhea Treatment

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasite Toxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals. Dehydroepiandrosterone (DHEA) has proved to be an important molecule that could drive resistance against a variety of infections, including intracellular parasites such as Plasmodium falciparum and Trypanozoma cruzi, among others. However, to date, the role of DHEA on T. gondii has not been explored. Here, we demonstrated for the first time the toxoplasmicidal effect of DHEA on extracellular tachyzoites. Ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading to the loss of the organelle structure and organization as well as the loss of the cellular shape. In vitro treatment with DHEA reduces the viability of extracellular tachyzoites and the passive invasion process. Two-dimensional (2D) SDS-PAGE analysis revealed that in the presence of the hormone, a progesterone receptor membrane component (PGRMC) with a cytochrome b5 family heme/steroid binding domain-containing protein was expressed, while the expression of proteins that are essential for motility and virulence was highly reduced. Finally, in vivo DHEA treatment induced a reduction of parasitic load in male, but not in female mice.

scholarly journals C-Terminal Domain of ABL Family Kinases, ABL and ARG, Defines Their Distinct Leukemogenic Activities in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2368-2368 ◽  
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Tsukimi Shoji ◽  
Taira Maekawa ◽  
Keiko Okuda
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Fusion Proteins ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Terminal Domain ◽  
C Terminus

Abstract ARG (ABL2) is a member of ABL family kinases and highly homologous to ABL (ABL1) except the C-terminal domain adjacent to the kinase domain. TEL/ARG that consists of ARG fused to TEL (ETV6) has been found in AML M3, M4 or T-ALL patients, with additional chromosomal abnormalities of t(15;17)(q12;q21), inv(16)(p13;q12) or t(1;10;12)(q25;q23;p13) translocation, respectively. The structure of TEL/ARG is similar to that of TEL/ABL, which has been found in patients with T-ALL, B-ALL, AML and CML. TEL mediates homo-oligomerization of these fusion proteins, TEL/ABL and TEL/ARG, resulting in constitutive activation of the tyrosine kinases. Although ABL fusion proteins such as BCR/ABL and TEL/ABL have been intensively investigated, the involvement of TEL/ARG in leukemogenesis is not fully elucidated yet. We have recently reported that in vitro transforming activity of TEL/ARG was significantly lower than that of TEL/ABL although their kinase activities were almost identical. Interestingly, the in vitro transforming activities of C-terminus-swapped mutants, TEL/ABL with C-terminal domain of ARG [TEL-ABL (ARG-C)] or TEL/ARG with C-terminal domain of ABL [TEL/ARG (ABL-C)], were comparable to those of TEL/ARG or TEL/ABL, respectively, while kinase activities in the swapped mutants were not altered. These results suggest that C-termini of ABL family kinases contain some functional domain that defines their distinct transforming activities. The purpose of this study is to compare the in vivo leukemogenic activities of TEL/ABL and TEL/ARG, and evaluate the impact of the C-terminal domains. First, we investigated whether TEL/ABL or TEL/ARG caused leukemia in mice. Each fusion gene together with GFP gene was retrovirally transduced into the bone marrow cells harvested from C57BL/6 mice treated with 5-fluorouracil, and the transduced cells were transplanted into lethally irradiated mice. Similar to BCR/ABL, transplantation of TEL/ABL-transduced cells induced rapid myeloproliferative status accompanied by hepatomegaly and/or splenomegaly, and all the recipient mice died within 33 days after transplantation, indicating the development of myeloid leukemia. In contrast, the recipient mice transplanted with TEL/ARG-transduced cells did not develop myeloid leukemia but infiltrative mastocytosis, and died around 200 days after transplantation (Figure 1). Hemophagocytic mast cells accumulating in the bone marrow, and mast cells circulating in the peripheral blood were also observed in these mice. Next we investigated the roles of C-terminal domains of ABL and ARG in their in vivo leukemogenic activities. C-terminus-swapped mutants, TEL/ABL (ARG-C) and TEL/ARG (ABL-C) were retrovirally transduced into bone marrow cells and the transduced cells were transplanted as described above. Intriguingly, TEL/ABL (ARG-C) mutant failed to cause myeloproliferative status or leukemia at day 153 (Figure 2A). On the other hand, TEL/ARG (ABL-C) induced lethal myeloid leukemia in 4 out of 13 mice (30.8%) within 111 days after transplantation (Figure 2B). Collectively, the in vivo phenotypes induced by TEL/ABL (ARG-C) or TEL/ARG (ABL-C) resembled those induced by TEL/ARG or TEL/ABL, respectively. Mastocytosis, a characteristic of TEL-ARG-induced phenotype, has not been observed so far in any of the recipients of TEL/ABL (ARG-C) or TEL/ARG (ABL-C). In conclusion, these results indicate that C-terminal domain of ABL family kinases defines their distinct leukemogenic activities in vivo through modulating both proliferation and differentiation. Notably, C-terminus of ARG strongly suppressed the in vivo leukemogenic activity of TEL/ABL without impairing the tyrosine kinase activity. Further clarification of the molecular mechanisms underlying the suppressive activity of C-terminus of ARG will lead to development of a novel therapeutic strategy, especially for patients with CML harboring mutations, which are resistant to tyrosine kinase inhibitors. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 2795-2804 ◽  
Author(s):  
Marcel van Lith ◽  
Anna-Riikka Karala ◽  
Dave Bown ◽  
John A. Gatehouse ◽  
Lloyd W. Ruddock ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Germ Cells ◽  
Specific Protein ◽  
Developmentally Regulated ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Disulfide Isomerases ◽  
Chaperone Complex

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

scholarly journals Molecular mechanisms of somatostatin receptor trafficking

2011 ◽  
Vol 48 (1) ◽  
pp. R1-R12 ◽  
Author(s):  
Zsolt Csaba ◽  
Stéphane Peineau ◽  
Pascal Dournaud
Keyword(s):  
Molecular Mechanisms ◽  
Somatostatin Receptor ◽  
Cell Types ◽  
Exocrine Secretion ◽  
Receptor Subtypes ◽  
Hydrophobic Core ◽  
Somatostatin Receptor Subtypes ◽  
C Terminus

The neuropeptide somatostatin (SRIF) is an important modulator of neurotransmission in the central nervous system and acts as a potent inhibitor of hormone and exocrine secretion. In addition, SRIF regulates cell proliferation in normal and tumorous tissues. The six somatostatin receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, and sst5), which belong to the G protein-coupled receptor (GPCR) family, share a common molecular topology: a hydrophobic core of seven transmembrane-spanning α-helices, three intracellular loops, three extracellular loops, an amino-terminus outside the cell, and a carboxyl-terminus inside the cell. For most of the GPCRs, intracytosolic sequences, and more particularly the C-terminus, are believed to interact with proteins that are mandatory for either exporting neosynthesized receptor, anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Accordingly, most of the SRIF receptors can traffic not onlyin vitrowithin different cell types but alsoin vivo. A picture of the pathways and proteins involved in these processes is beginning to emerge.

scholarly journals Intramolecular interactions between the AF3 domain and the C-terminus of the human progesterone receptor are mediated through two LXXLL motifs

2004 ◽  
Vol 32 (3) ◽  
pp. 843-857 ◽  
Author(s):  
X Dong ◽  
SJ Lye ◽  
Keyword(s):  
Progesterone Receptor ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Direct Interaction ◽  
Intramolecular Interactions ◽  
Dependent Manner ◽  
C Terminus ◽  
Human Progesterone Receptor

The human progesterone receptor (PR) exists in two major forms, PRA and PRB, which differentially regulate gene transcription in a cell- and promoter-specific manner. The molecular mechanisms underlying this differential transcriptional activity have been attributed to the presence of a unique AF3 domain within PRB that may result in the two isoforms adopting different protein conformations. We demonstrate here that in myometrial cells, PRB exhibits strong progesterone-dependent transcriptional activity that is dependent on the presence of two LXXLL motifs within the AF3 domain. In vitro and in vivo protein interaction assays indicate that these motifs mediate the direct interaction between the AF3 domain and C-PR in a progesterone-dependent manner. Mutation of either of the LXXLL motifs or deletion of the last 30 amino acids within the C-terminus disrupts this interaction and progesterone-dependent transcriptional activity of PRB. Members of the p160 family of co-activators (such as GRIP-1) also interact with C-PR through their LXXLL motifs. However, GRIP-1 does not compete with AF3 but rather acts to synergize these two transactivation domains. Our data suggest that a failure to form an appropriate AF3-C-terminus interaction results in an inability of co-activators to induce maximal PR-dependent transactivation. The absence of an AF3 domain within PRA may account for its inability to activate progesterone-responsive genes, as well as its actions as a dominant trans-repressor.

scholarly journals Inhibition of Secretion by 1,3-Cyclohexanebis(methylamine), a Dibasic Compound That Interferes with Coatomer Function

1999 ◽  
Vol 10 (4) ◽  
pp. 921-933 ◽  
Author(s):  
Tonghuan Hu ◽  
Chia-Yi Kao ◽  
Robert Tod Hudson ◽  
Alice Chen ◽  
Rockford K. Draper
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Amino Groups ◽  
Intact Cells ◽  
Golgi Membranes ◽  
Lysine Residues ◽  
Full Complement ◽  
Primary Amino

We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the ε-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1,3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane.

scholarly journals GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Xiaolin Li ◽  
Sixuan Liu ◽  
Xuan Chen ◽  
Run Huang ◽  
Lisi Ma ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Ovarian Function ◽  
Mtor Signaling ◽  
Unfolded Protein ◽  
Hormone Analogs

AbstractChemotherapy-induced ovarian dysfunction is a serious adverse effect in premenopausal patients with cancer. Gonadotrophin-releasing hormone analogs (GnRHa) protect ovarian function, but its molecular mechanisms have not yet been determined. In this study, we attempted to determine the previously unknown molecular mechanism by which such protection occurs. Serum anti-Müllerian hormone (AMH) levels were tested in tumor-bearing nude mice, a series of exploratory experiments were conducted. We discovered that GnRHa protects granulosa cells from chemotherapeutic toxicity in vivo and in vitro. We also showed that CTX-induced endoplasmic reticulum stress inhibits the secretion of AMH, and treatment with GnRHa relieves ER stress and the subsequent unfolded-protein response by modulating mTOR signaling to induce autophagy. The results of mechanistic studies indicated that GnRHa-modulated mTOR signaling to induce autophagy, which alleviated CTX-induced ER stress and promoted the secretion of AMH.

scholarly journals Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways

2001 ◽  
Vol 12 (8) ◽  
pp. 2482-2496 ◽  
Author(s):  
Nica Borgese ◽  
Ilaria Gazzoni ◽  
Massimo Barberi ◽  
Sara Colombo ◽  
Emanuela Pedrazzini
Keyword(s):  
Endoplasmic Reticulum ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mitochondrial Outer Membrane ◽  
C Terminus ◽  
Green Fluorescent ◽  
Ta Proteins

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b 5, a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.

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