Effect of Polyphosphorylation on Behavior of Protein Disordered Regions.

Proteins interact with many charged biological macromolecules (polyelectrolytes), including inorganic polyphosphates. Recently a new protein post-translational modification, polyphosphorylation, or a covalent binding of polyphosphate chain to lysine, was demonstrated in human and yeast. Herein, we performed the first molecular modeling study of a possible effect of polyphosphorylation on behavior of the modified protein using replica exchange molecular dynamics simulations in atomistic force field with explicit water. Human endoplasmin (GRP-94), a member of heat shock protein 90 family, was selected as a model protein. Intrinsically disordered region in N-terminal domain serving as a charged linker between domains and containing a polyacidic serine and lysine-rich motif, was selected as a potent polyphosphorylation site according to literature data. Polyphosphorylation, depending on exact modification site, has been shown to influence on the disordered loop flexibility and induce its further expanding, as well as induce changes in interaction with ordered part of the molecule. As a result, polyphosphorylation in N-terminal domain might affect interaction of HSP90 with client proteins since these chaperones play a key role in protein folding.

NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90.

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90ß, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

Proteostasis and ageing: insights from long-lived mutant mice.

The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis - where proteostasis is defined here as the maintenance of the proteome - appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants.

Proteomic analysis of the secretome of equine herpesvirus-1 infected rabbit kidney cells.

Herpesviruses are the main cause of abortions and respiratory or neurological disorders in horses. Various disease patterns are suspected to be associated with the A2254G point mutation in the DNA polymerase sequence (ORF30) of the herpesvirus genome, although the importance of this link is still under debate. Based on a label-free quantitative proteomic analysis, the differences in the secretion of some host proteins between rabbit kidney cells infected with A2254 and cells of the same line infected with G2254 equine herpesvirus 1 (EHV-1) strains were identified. In both groups, downregulation of proteins involved in insulin growth factor and extracellular matrix pathway regulation was observed. Among 12 proteins with increased secretion, 8 were regulated only in G2254 EHV-1 infection. Those were endoplasmic reticulum chaperones with calcium binding properties, related to unfolded protein response and mitochondria. It was presumed that the secretion of proteins such as calreticulin, Hspa5 or endoplasmin may contribute to the pathogenesis of EHV-1 infection.

Expression of CLAVATA3 fusions indicates rapid intracellular processing and a role of ERAD.

The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum chaperone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

Quantitative proteomics and biochemical analyses reveal the role of endoplasmin in the regulation of the expression and secretion of A Disintegrin And Metalloproteinase 12.

A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in multiple cancers such as breast and cervical cancers and its high expression reduces the overall patient survival rate. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently in cells are not clear. Here, we use affinity purification and mass spectrometry to identify the ADAM12S-interacting proteins. Spectral counting and MaxQuant label-free quantification reveal that ADAM12S but not ADAM12L specifically interacts with a subset of endoplasmic reticulum proteins, such as endoplasmin (GRP94), 78 kDa glucose-regulated protein (GRP78), and UDP-glucose:glycoprotein glucosyltransferase I (UGGT1), that regulate the folding and processing of secreted proteins. Further biochemical experiments validate the interaction between ADAM12S and several of its interacting proteins. Computational docking analysis demonstrates that GRP94 preferentially interacts with ADAM12S over ADAM12L. The data also suggest that both the protein expression level and the secretion of ADAM12S are regulated by GRP94 expression and knockdown. Our results reveal a link between these two proteins that are highly expressed in cancer cells. Furthermore, our studies define a new ADAM12S-specific regulator that may contribute to the cancer development. SIGNIFICANCE: A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in many cancers such as lung, breast, and cervical cancers. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently are not completely understood. We use affinity purification and label-free quantitative proteomics to identify the ADAM12S-interacting proteins. Our results reveal that ADAM12S specifically interacts with a subset of endoplasmic reticulum proteins, including endoplasmin (GRP94), UDP-glucose:glycoprotein glucosyltransferase I (UGGT1), and neutral α-glucosidase AB (GANAB). Computer modeling reveals that ADAM12S interacts with the surface amino acids of GRP94 more strongly than ADAM12L. Biochemical experiments further reveal that GRP94 regulates both the protein level and the secretion of ADAM12S. Database mining finds that both GRP94 and ADAM12 are highly expressed in multiple cancers and their high expression is correlated with poor patient survival rate. Taken together, our work discovers a new upstream regulator for ADAM12S, which may contribute to its distinct functions in the regulation of the migration and invasion of cancer cells.

Molecular cloning, cellular expression and characterization of Arabian camel (Camelus dromedarius) endoplasmin.

Endoplasmin, or GRP94, is an ER-located stress inducible molecular chaperone implicated in the folding and assembly of many proteins. The Arabian one-humped camel lives in an environment of thermal stress, nevertheless is able to encounter the risk of misfolded proteins. Here, the cDNA encoding camel GRP94 was isolated by rapid amplification of cDNA ends. The isolated cDNA contained an open reading frame of 2412 bp encoding a protein of 803 amino acids with predicted molecular mass of 92.5 kDa. Nucleotide and protein BLAST analysis of cGRP94 revealed strong conservation between camel and other domestic mammals. Overexpression of cGRP94 in COS-1 cells revealed multiple isoforms including one N-glycosylated species. Immunofluorescence colocalized cGRP94 with the ER resident protein calnexin. Interestingly, none of the cGRP94 isoforms expressed in CHO cells was N-glycosylated, presumably due to folding determinants that mask the N-glycosylation sites as proposed by in silico modelling. Surprisingly, isoforms of cGRP94 were detected in the culture media of transfected cells indicating that the protein, although an ER resident, also is trafficked and secreted into the exterior milieu. The overall striking structural homologies of GRP94s among mammalian reflect their pivotal role in the ER quality control and protein homeostasis.

Calcium-dependent protein folding in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by a progressive loss of motor neurons. Although the etiology remains unclear, disturbances in Ca2+ homoeostasis and protein folding are essential features of neurodegeneration. The correct folding of proteins is managed by folding proteins, which are regulated by Ca2+ levels. Therefore, Ca(2+)-sensitive folding proteins represent an important link between disturbed Ca2+ handling and protein misfolding in amyotrophic lateral sclerosis. In the first part of this review, we focus on Ca2+ handling in the endoplasmic reticulum and mitochondria in terms of their roles in protein misfolding. In the second part, we draw attention to the main Ca(2+)-sensitive folding proteins that play a role in motor neuron degeneration such as calreticulin and calnexin, which are involved in the folding of glycosylated proteins. In addition, calmodulin and the Ca2+/calmodulin-dependent protein kinase are discussed as one correlation to oxidative stress. The heat shock protein endoplasmin is associated with the anti-apoptotic insulin-like growth factor pathway that is altered in amyotrophic lateral sclerosis. Grp78, which influences Ca2+ homeostasis in the intraluminal endoplasmic reticulum is upregulated in mice models and amyotrophic lateral sclerosis patients and constitutes a core component of the unfolded protein response. Lastly, the protein disulfide isomerase family is responsible for mediating oxidative protein folding in the endoplasmic reticulum.

Expression of ERp29 in model of Parkinson's disease of PC12 cells induced by proteasome inhibitor / 吉林大学学报(医学版)

Objective To investigate the pathogenetic mechanism of ubiquitin-proteasome dysfunction in a model of Parkinson's disease(PD),which can provide the theoretical basis for PD.Methods After establishment of PD model induced by PSI in PC12 cells,proteins of untreated(DMSO) and PSI-treated PC12 cells were extracted 36 h after incubation,and then the maps of the extracted proteins were established by DIGE system.The altered protein spots were identified with MALDI-TOF Pro MS and database searching.Results Thirty-six treatment of PC12 cells with PSI induced the appearance of cytoplasmic Lewy body-like eosinophilic inclusions and apoptosis.The percentage of apoptotic cells was 25.53%.ERp29 were identified by MALDITOF Pro MS.The expression of ERp29 decreased in treatment group,compared with normal group(P