scholarly journals Proteolytic processing of QSOX1A ensures efficient secretion of a potent disulfide catalyst

2013 ◽  
Vol 454 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Jana Rudolf ◽  
Marie A. Pringle ◽  
Neil J. Bulleid
Keyword(s):  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Subcellular Location ◽  
Proteolytic Processing ◽  
Protein Substrates ◽  
Wide Range ◽  
Efficient Processing ◽  
Quiescin Sulfhydryl Oxidase

QSOX1 (quiescin sulfhydryl oxidase 1) efficiently catalyses the insertion of disulfide bonds into a wide range of proteins. The enzyme is mechanistically well characterized, but its subcellular location and the identity of its protein substrates remain ill-defined. The function of QSOX1 is likely to involve disulfide formation in proteins entering the secretory pathway or outside the cell. In the present study, we show that this enzyme is efficiently secreted from mammalian cells despite the presence of a transmembrane domain. We identify internal cleavage sites and demonstrate that the protein is processed within the Golgi apparatus to yield soluble enzyme. As a consequence of this efficient processing, QSOX1 is probably functional outside the cell. Also, QSOX1 forms a dimer upon cleavage of the C-terminal domain. The processing of QSOX1 suggests a novel level of regulation of secretion of this potent disulfide catalyst and producer of hydrogen peroxide.

scholarly journals Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

1986 ◽  
Vol 6 (9) ◽  
pp. 3191-3199 ◽  
Author(s):  
C J Langford ◽  
S J Edwards ◽  
G L Smith ◽  
G F Mitchell ◽  
B Moss ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Poor Response ◽  
Subcellular Location ◽  
Epidemiological Data ◽  
Recombinant Vaccinia Virus ◽  
Live Virus ◽  
Recombinant Vaccinia ◽  
Wide Range ◽  
Vaccinia Viruses ◽  
Infected Cells

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity

1986 ◽  
Vol 6 (9) ◽  
pp. 3191-3199
Author(s):  
C J Langford ◽  
S J Edwards ◽  
G L Smith ◽  
G F Mitchell ◽  
B Moss ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Poor Response ◽  
Subcellular Location ◽  
Epidemiological Data ◽  
Recombinant Vaccinia Virus ◽  
Live Virus ◽  
Recombinant Vaccinia ◽  
Wide Range ◽  
Vaccinia Viruses ◽  
Infected Cells

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

scholarly journals Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

2003 ◽  
Vol 14 (3) ◽  
pp. 1268-1278 ◽  
Author(s):  
Laurence Fayadat ◽  
Ron R. Kopito
Keyword(s):  
Quality Control ◽  
Plasma Membrane ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Proteasome Inhibitors ◽  
Integral Membrane Protein ◽  
Type I ◽  
Α Subunit ◽  
Influenza Hemagglutinin

To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.

scholarly journals Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

1997 ◽  
Vol 8 (10) ◽  
pp. 1943-1954 ◽  
Author(s):  
Jian-Xin Zhang ◽  
Ineke Braakman ◽  
Kent E.S. Matlack ◽  
Ari Helenius
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Influenza Hemagglutinin ◽  
Culture Cells ◽  
Folded Structure ◽  
Low Concentrations ◽  
Sorting Process

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.

scholarly journals Two phases of disulfide bond formation have differing requirements for oxygen

2013 ◽  
Vol 203 (4) ◽  
pp. 615-627 ◽  
Author(s):  
Marianne Koritzinsky ◽  
Fiana Levitin ◽  
Twan van den Beucken ◽  
Ryan A. Rumantir ◽  
Nicholas J. Harding ◽  
...  
Keyword(s):  
Er Stress ◽  
Disulfide Bond ◽  
Electron Acceptor ◽  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Terminal Electron Acceptor ◽  
Two Phases

Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress.

scholarly journals Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels

2001 ◽  
Vol 114 (21) ◽  
pp. 3845-3855 ◽  
Author(s):  
Patricia E. M. Martin ◽  
Geraldine Blundell ◽  
Shoeb Ahmad ◽  
Rachel J. Errington ◽  
W. Howard Evans
Keyword(s):  
Gap Junctions ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Fluorescent Proteins ◽  
Alternative Pathway ◽  
Brefeldin A ◽  
Junctional Communication ◽  
In Cells

The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.

scholarly journals Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface

2010 ◽  
Vol 430 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Marit Stirnberg ◽  
Eva Maurer ◽  
Angelika Horstmeyer ◽  
Sonja Kolp ◽  
Stefan Frank ◽  
...  
Keyword(s):  
Cell Surface ◽  
Disulfide Bonds ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Bond Cleavage ◽  
Proteolytic Processing ◽  
Cleavage Sites ◽  
Single Chain

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

2017 ◽  
Author(s):  
Jana Shen ◽  
Zhi Yue ◽  
Helen Zgurskaya ◽  
Wei Chen
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Proton Release ◽  
Intrinsic Resistance ◽  
Constant Ph ◽  
Wide Range ◽  
Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

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