Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a mechanism in which the sulfhydryl group of a cysteine residue at the active center acts as a nucleophile.
The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b(+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.
We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
Activated CD8(+) T cells discriminate infected and tumor cells from normal self by recognizing MHC class I-bound peptides on the surface of antigen-presenting cells. The mechanism by which MHC class I molecules select optimal peptides against a background of prevailing suboptimal peptides and in a considerably proteolytic ER environment remained unknown. Here, we identify protein disulfide isomerase (PDI), an enzyme critical to the formation of correct disulfide bonds in proteins, as a component of the peptide-loading complex. We show that PDI stabilizes a peptide-receptive site by regulating the oxidation state of the disulfide bond in the MHC peptide-binding groove, a function that is essential for selecting optimal peptides. Furthermore, we demonstrate that human cytomegalovirus US3 protein inhibits CD8(+) T cell recognition by mediating PDI degradation, verifying the functional relevance of PDI-catalyzed peptide editing in controlling intracellular pathogens. These results establish a link between thiol-based redox regulation and antigen processing.
Evidence
2:
Inferred from Physical InteractionIntAct
The beta-amyloids (abetas) are the major components of the plaque observed in the brains of patients with Alzheimer's disease. The conundrum is that although they are produced in everyone during the posttranslational processing in the endoplasmic reticulum (ER) of the amyloid precursor protein (APP), deposits are only observed in the elderly. Our work suggests that normals have a carrier protein(s) keeping them in solution. Based on immunoblotting studies of cerebrospinal fluid (CSF) from normals, we find that the bulk of the abetas are bound to the ER chaperones, ERp57 and calreticulin, suggesting that these may be carrier proteins which prevent aggregation of the abetas and that the deposits are due to faulty ER posttranslational processing of APP with the failure to form this complex. If membrane protein synthesis is similarly affected, it could explain the neuronal dysfunction characteristic of Alzheimer's disease.
The function of a 58-kDa liver microsomal protein (P58) is controversial. To help clarify the physiological function of this protein, particularly in humans, a full-length human liver cDNA clone was isolated, sequenced, and expressed in milligram quantities with the use of a baculovirus expression system. The deduced amino acid sequence of the mature protein contained two thioredoxin-like active site motifs (CGHC) and in its C-terminus a nuclear localization motif (KPKKKKK), and an ER-retention/retrieval motif (QEDL). The mature form of human P58 shared 95% amino acid sequence identity with the deduced amino acid sequences of a bovine liver cDNA, 93% with a murine B lymphocyte cDNA, and 91% with a rat basophilic leukemia cell cDNA. In contrast to reports on the activities of nonhuman forms of P58, the purified expressed human P58 showed no carnitine acyltransferase or protease activities. However, it did have protein disulfide isomerase activity, indicating that the physiological activity of human liver P58 may be attributed, at least in part, to this activity.
The retention in the endoplasmic reticulum (ER) lumen of soluble resident proteins. Sorting receptors retrieve proteins with ER localization signals, such as KDEL and HDEL sequences or some transmembrane domains, that have escaped to the cis-Golgi network and return them to the ER. Abnormally folded proteins and unassembled subunits are also selectively retained in the ER.
The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b(+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.
The function of a 58-kDa liver microsomal protein (P58) is controversial. To help clarify the physiological function of this protein, particularly in humans, a full-length human liver cDNA clone was isolated, sequenced, and expressed in milligram quantities with the use of a baculovirus expression system. The deduced amino acid sequence of the mature protein contained two thioredoxin-like active site motifs (CGHC) and in its C-terminus a nuclear localization motif (KPKKKKK), and an ER-retention/retrieval motif (QEDL). The mature form of human P58 shared 95% amino acid sequence identity with the deduced amino acid sequences of a bovine liver cDNA, 93% with a murine B lymphocyte cDNA, and 91% with a rat basophilic leukemia cell cDNA. In contrast to reports on the activities of nonhuman forms of P58, the purified expressed human P58 showed no carnitine acyltransferase or protease activities. However, it did have protein disulfide isomerase activity, indicating that the physiological activity of human liver P58 may be attributed, at least in part, to this activity.
Enzyme that catalyzes the 1,1-, 1,2- or 1,3-hydrogen shift. The 1,1- hydrogen shift is an inversion at an asymmetric carbon center (racemases, epimerases). The 1,2-hydrogen shift involved a hydrogen transfer between two adjacent carbon atoms, one undergoing oxidation, the other reduction (aldose-ketose isomerases). The 1,3-hydrogen shifts are allylic or azaallylic (when nitrogen is one of the three atoms) isomerizations.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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