In vitro folding of methionine-arginine human lyspro-proinsulin S-sulfonate-Disulfide formation pathways and factors controlling yield

2010 ◽  
Vol 26 (5) ◽  
pp. 1332-1343 ◽  
Author(s):  
Shuang Chen ◽  
Lawrence Adijanto ◽  
Nien-Hwa Linda Wang
Keyword(s):  
Disulfide Formation

scholarly journals Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p

2013 ◽  
Vol 432 (3) ◽  
pp. 438-443 ◽  
Author(s):  
Siti N.H. Mohd Yusuf ◽  
Ulla-Maja Bailey ◽  
Nikki Y. Tan ◽  
Muhammad Fairuz Jamaluddin ◽  
Benjamin L. Schulz
Keyword(s):  
Disulfide Formation ◽  
Mixed Disulfide

scholarly journals Activation of cell-penetrating peptide fragments by disulfide formation

Amino Acids ◽  
2020 ◽  
Vol 52 (8) ◽  
pp. 1161-1168
Author(s):  
Raheleh Tooyserkani ◽  
Wojciech Lipiński ◽  
Bob Willemsen ◽  
Dennis W. P. M. Löwik
Keyword(s):  
Targeted Drug Delivery ◽  
Disulfide Bridge ◽  
Cell Penetrating Peptides ◽  
Cysteine Residue ◽  
Fluorescent Label ◽  
Peptide Fragments ◽  
Disulfide Formation ◽  
Cell Penetrating ◽  
Uptake Activity

Abstract Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery.

scholarly journals Distinct roles and actions of PDI family enzymes in catalysis of nascent-chain disulfide formation

2021 ◽  
Author(s):  
Chihiro Hirayama ◽  
Kodai Machida ◽  
Kentaro Noi ◽  
Tadayoshi Murakawa ◽  
Masaki Okumura ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Single Molecule ◽  
Disulfide Bonds ◽  
High Speed ◽  
Binding Pocket ◽  
Face To Face ◽  
Disulfide Formation ◽  
Nascent Chain ◽  
Single Molecule Analysis

AbstractThe mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains (RNC) of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with short segments exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Similarly to ERp46, a PDI mutant with an occluded substrate-binding pocket displayed shorter-time RNC binding and higher efficiency in disulfide introduction than wild-type PDI. Altogether, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation in RNC when longer nascent chains emerge out from ribosome.

Abstract 806: Protein Disulfide Isomerase (PDI) signals Tissue Factor (TF)-dependent Fibrin formation in vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Marie-Luise von Bruehl ◽  
Lenka Grahl ◽  
Ildiko Konrad ◽  
Michael Lorenz ◽  
Christian Schulz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Arterial Thrombosis ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Intravascular Coagulation ◽  
Disulfide Formation ◽  
Fibrin Formation ◽  
Protein Disulfide

Background: Pathological fibrin formation is a predominant cause for arterial thrombosis, triggering myocardial infarction and stroke. Tissue factor (TF), which is expressed by vascular endothelium and cell-derived microparticles, is a central trigger of intravascular fibrin generation. In intact blood vessels, TF is only minimally active, and the coagulation system is down-regulated. In contrast, TF is rapidly stimulated following endothelial disruption. However, the molecular mechanisms that trigger fibrin formation via regulation of TF activation are largely undefined. Methods and Results: Here, we have identified a novel pathway that leads to TF activation in vitro and contributes to intravascular coagulation in vivo. We show that the protein disulfide isomerase (PDI) induces disulfide formation of the Cys186/Cys209 pair located in the extracellular domain of TF. In vitro, PDI-mediated disulfide formation led to profound activation of TF. In vivo, we found that PDI is not present in the intact vessel wall, but becomes abundantly expressed following vessel damage. We show that injured smooth muscle cells and adherent/activated platelets are the major cellular sources of PDI at sites of vascular injury. We then assessed fibrin formation following injury of the common carotid artery using intravital microscopy. We observed that PDI-blockade strongly reduced fibrin formation by approximately 45– 60%. Likewise, intravenous infusion of PDI significantly enhanced TF-dependent fibrin formation. Together, this implies that PDI initiates coagulation in vitro and also during arterial thrombosis in vivo. Notably, PDI did not act on platelets to trigger intravascular coagulation. Correspondingly, PDI blockade also inhibited fibrin formation, when platelet adhesion was prevented using a function-blocking anti-GPVI antibody. Conclusion: These findings reveal for the first time in vivo that the thiol isomerase PDI is a major molecular trigger of blood coagulation, which is exposed after vessel injury and converts TF into its functionally active form. Hence, PDI represents a promising novel target for the treatment of pathologic fibrin formation in patients with thrombotic disorders.

scholarly journals The Paralogous MarR/DUF24-Family Repressors YodB and CatR Control Expression of the Catechol Dioxygenase CatE in Bacillus subtilis

2010 ◽  
Vol 192 (18) ◽  
pp. 4571-4581 ◽  
Author(s):  
Bui Khanh Chi ◽  
Kazuo Kobayashi ◽  
Dirk Albrecht ◽  
Michael Hecker ◽  
Haike Antelmann
Keyword(s):  
Bacillus Subtilis ◽  
Redox Regulation ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Binding Assays ◽  
Catechol Dioxygenase ◽  
Disulfide Formation ◽  
Redox Sensing

ABSTRACT The redox-sensing MarR/DUF24-type repressor YodB controls expression of the azoreductase AzoR1 and the nitroreductase YodC that are involved in detoxification of quinones and diamide in Bacillus subtilis. In the present paper, we identified YodB and its paralog YvaP (CatR) as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Inactivation of both CatR and YodB is required for full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N5-GTAA overlapping the −35 promoter region (BS1) and the transcriptional start site (TSS) (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue, which is required for redox sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data suggest that CatR is modified by intermolecular disulfide formation in response to diamide and quinones in vitro and in vivo. Redox regulation of CatR occurs independently of YodB, and no protein interaction was detected between CatR and YodB in vivo using protein cross-linking and mass spectrometry.

scholarly journals In vitro studies on the metabolic pathway of SQ 14225 (captopril) and mechanism of mixed disulfide formation.

1981 ◽  
Vol 4 (9) ◽  
pp. 677-684 ◽  
Author(s):  
TORU KOMAI ◽  
TOSHIHIKO IKEDA ◽  
KENJI KAWAI ◽  
EMI KAMEYAMA ◽  
HIDEYO SHINDO
Keyword(s):  
Metabolic Pathway ◽  
In Vitro Studies ◽  
Disulfide Formation ◽  
Mixed Disulfide

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

1978 ◽  
Vol 36 (2) ◽  
pp. 88-89
Author(s):  
M. Kraemer ◽  
J. Foucrier ◽  
J. Vassy ◽  
M.T. Chalumeau
Keyword(s):  
Plasma Proteins ◽  
Rat Hepatocytes ◽  
Ultrastructural Localization ◽  
Carrier Proteins ◽  
Normal Cells ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Monospecific Antisera ◽  
Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

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