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1.
Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long‐lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N‐terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser‐ and Thr‐containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N‐terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co‐ordinates zinc and that once zinc is co‐ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long‐lived proteins, for example truncation on the N‐terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long‐term stability of cellular proteins.  相似文献   

2.
3.
Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.  相似文献   

4.
Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO2·nH2O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N‐terminal peptides were not identified. We found the sequences of these N‐terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4‐bromine‐phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N‐terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.  相似文献   

5.
Racemization is one of the most abundant modifications in long‐lived proteins. It has been proposed that the accumulation of such modifications over time could lead to changes in tissues and ultimately human age‐related diseases. Serine is one of the main amino acids involved in racemization; however, the site of D‐Ser in any aged protein has yet to be reported. In this study, racemization of two residues, Ser 59 and Ser 62, has been demonstrated in an unstructured region of the small heat shock protein, αA‐crystallin. αA‐crystallin is also the most abundant structural protein in the human lens. D‐Ser increased linearly with age in normal lenses, until it accounted for approximately 35% of the Ser at both sites by the age of 75 years. In agreement with a possible role in human age‐related disease, levels were significantly higher in cataract lenses. It is likely that such prevalent age‐related changes contribute to the denaturation of α‐crystallin, and therefore its ability to act as a chaperone. Racemization of amino acids, such as serine, in flexible regions of long‐lived proteins, could be associated with the development of human age‐related conditions such as cataract.  相似文献   

6.
Ball LE  Garland DL  Crouch RK  Schey KL 《Biochemistry》2004,43(30):9856-9865
Because of the lack of protein turnover in fiber cells of the ocular lens, Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, undergoes extensive post-translational modification with fiber cell age. To map the distribution of modified forms of AQP0 within the lens, normal human lenses ranging in age from 34 to 38 were concentrically dissected into several cortical and nuclear sections. Membrane proteins still embedded in the membranes were digested with trypsin, and the resulting C-terminal peptides of AQP0 were analyzed by HPLC tandem mass spectrometry, permitting the identification of modifications and estimation of their abundance. Consistent with earlier reports, the major phosphorylation site was Ser 235, and the major sites of backbone cleavage occurred at residues 246 and 259. New findings suggest that cleavage at these sites may be a result of nonenzymatic truncation at asparagine residues. In addition, this approach revealed previously undetected sites of truncation at residues 249, 260, 261, and 262; phosphorylation at Ser 231 and to a lower extent at Ser 229; and racemization/isomerization of l-Asp 243 to d-Asp and d-iso-Asp. The spatial distribution of C-terminally modified AQP0 within the lens indicated an increase in truncation and racemization/isomerization with fiber cell age, whereas the level of Ser 235 phosphorylation increased from the outer to inner cortex but decreased in the nucleus. Furthermore, the remarkably similar pattern and distribution of truncation products from lenses from three donors suggest specific temporal mechanisms for the modification of AQP0.  相似文献   

7.
Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.  相似文献   

8.
The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy-terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an Mr of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.  相似文献   

9.
alphaA-crystallin is a major protein component of the human lens. It is known to undergo posttranslational modification. This study was done to further elucidate the temporal and spatial nature of these posttranslational modifications and to correlate the modified forms with electrophoretic migration. We dissected normal human lenses into concentric shells of fiber cells, separated the proteins by two-dimensional electrophoresis, and identified modified forms by mass spectrometry. We found that alphaA-crystallin migrated as a major spot and in over 20 additional protein spots. The extent of modification correlated with the age of the fiber cells and the depth within a lens. A correlation was also seen between these parameters and the concentration of modified forms that had full-length sequences but migrated at more acidic positions. These proteins were phosphorylated, acetylated, and/or deamidated. A few proteins migrated to a more basic position than the major form of alphaA-crystallin. The locations of several species that were truncated after C-terminal residues Ser172 and Ser162 were identified. Each of these species had intact N termini. The similarity of the C-terminal cleavage sites found in alphaA- and alphaB-crystallins was noted.  相似文献   

10.
The amino acid sequences of apolipoprotein E (apoE) from 63 different mammalian species have been downloaded from the protein database. The sequences were compared to human apoE4 to determine conserved and non‐conserved sequences of amino acids. ApoE4 is the major risk factor for the development of late onset Alzheimer's disease while apoE3, which differs from apoE4 by a single amino acid change at position 112, poses little or no risk for the development of this disease. Thus, the two proteins appear to be structurally and functionally different. Seven highly conserved regions, representing approximately 47 amino acids (of 299) have been found. These regions are distributed throughout the protein and reflect ligand binding sites as well as regions proposed to be involved in the propagation of the cysteine–arginine change at position 112 to distant regions of the protein in the N‐ and C‐terminal domains. Highly non‐conserved regions are at the N‐ and C‐terminal ends of the apoE protein.  相似文献   

11.
The calpain family of Ca2+‐dependent cysteine proteases plays a vital role in many important biological processes which is closely related with a variety of pathological states. Activated calpains selectively cleave relevant substrates at specific cleavage sites, yielding multiple fragments that can have different functions from the intact substrate protein. Until now, our knowledge about the calpain functions and their substrate cleavage mechanisms are limited because the experimental determination and validation on calpain binding are usually laborious and expensive. In this work, we aim to develop a new computational approach (LabCaS) for accurate prediction of the calpain substrate cleavage sites from amino acid sequences. To overcome the imbalance of negative and positive samples in the machine‐learning training which have been suffered by most of the former approaches when splitting sequences into short peptides, we designed a conditional random field algorithm that can label the potential cleavage sites directly from the entire sequences. By integrating the multiple amino acid features and those derived from sequences, LabCaS achieves an accurate recognition of the cleave sites for most calpain proteins. In a jackknife test on a set of 129 benchmark proteins, LabCaS generates an AUC score 0.862. The LabCaS program is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/LabCaS . Proteins 2013. © 2012 Wiley Periodicals, Inc.  相似文献   

12.
Liang GZ  Li SZ 《Biopolymers》2007,88(3):401-412
Factor analysis scales of generalized amino acid information (FASGAI) involving hydrophobicity, alpha and turn propensities, bulky properties, compositional characteristics, local flexibility, and electronic properties were derived from 516 property parameters of 20-coded amino acids, and was then employed to represent sequence structures of 746 peptides with 8 amino acid residues. Cleavage site prediction models for human immunodeficiency virus type 1 protease by linear discriminant analysis and support vector machine with radial basis function kernel were constructed to identify if they could be cleaved or not, and were further utilized to investigate the cleavage specificity. These diversified properties, including the bulky properties, secondary conformation characteristics, electronic properties, and hydrophobicity at the first, the second, the fourth, the fifth, and the sixth residue, are possibly important factors in determining HIV PR cleavage or not. Particularly, maximal positive and negative influences result from the bulky properties of different sites. Further results from analysis of variance also likely reflect that the HIV PR recognizes diversified key properties of various sites in the octameric sequences. Satisfactory results show that FASGAI can not only be used to represent sequence structures of various functional peptides, but alsoprovide a potential feasible measure for exploring relationship between protein motif sequences and their functions.  相似文献   

13.
The amino acid sequence of the N-terminal two-thirds of a trout high-mobility-group protein, HMG-T, has been determined as a continuous sequence of 174 residues out of a total for the whole molecule of 260 residues. When this sequence was compared with published sequences of long cyanogen bromide-derived peptides from the analogous calf-thymus proteins, HMG-1 and -2 (Walkeret al. , 1979), there was strong homology, with 60–70% identity of corresponding amino acid residues in the three proteins, the majority in lengthy identical runs. However, a discrepancy in the position of a highly acidic run of aspartic and glutamic residues suggests this region may not lie within the main polypeptide chain but may represent a separate chain or possibly a branched structure,  相似文献   

14.
Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit–subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.  相似文献   

15.
Characterization of aggrecan core protein peptides appearing in the medium of adult articular cartilage maintained in tissue culture showed that eight major peptides could be detected. The two largest peptides had the same N-terminal sequence as bovine aggrecan core protein and probably represent partly degraded aggrecan lost to the medium in the form of the proteoglycan aggregate. The three next smallest peptides were all shown to have another N-terminal sequence which corresponded to a sequence in the interglobular domain starting at alanine residue 393 of the human aggrecan core protein (K. Doege et al., 1991, J. Biol. Chem. 266, 894-902). Two other peptides were isolated and shown to have two different N-terminal amino sequences corresponding to sequences in the chondroitin sulfate attachment domain 2 of the core protein starting at alanine residue 1839 and leucine residue 1939 of human aggrecan. This suggests that the catabolism of aggrecan by adult articular cartilage occurs by the proteolytic cleavage of the core protein of this proteoglycan at three separate sites. Examination of the amino acid sequences around each of these cleavage sites showed a similar pattern TEGE decreases ARGS, TAQE decreases AGEG, and VSQE decreases LGQR, suggesting that a single proteinase may be involved in the catabolism of aggrecan. Analysis of synovial fluids and serum of age-matched animals revealed the presence of aggrecan core protein peptides corresponding in size to those detected in vitro, thus indicating the cleavage observed in explant culture is the same as that which occurs in vivo.  相似文献   

16.
Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.  相似文献   

17.
In Escherichia coli, the ClpAP protease, together with the adaptor protein ClpS, is responsible for the degradation of proteins bearing an amino‐terminal destabilizing amino acid (N‐degron). Here, we determined the three‐dimensional structures of ClpS in complex with three peptides, each having a different destabilizing residue—Leu, Phe or Trp—at its N terminus. All peptides, regardless of the identity of their N‐terminal residue, are bound in a surface pocket on ClpS in a stereo‐specific manner. Several highly conserved residues in this binding pocket interact directly with the backbone of the N‐degron peptide and hence are crucial for the binding of all N‐degrons. By contrast, two hydrophobic residues define the volume of the binding pocket and influence the specificity of ClpS. Taken together, our data suggest that ClpS has been optimized for the binding and delivery of N‐degrons containing an N‐terminal Phe or Leu.  相似文献   

18.
The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.  相似文献   

19.
Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln178 in HcPE‐1. The Arg residues (HcPE‐1, Arg132; HcPE‐2, Arg134; HcPE‐3, Arg325) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.  相似文献   

20.
The N‐end rule relates the in vivo half‐life of a protein to the identity of its N‐terminal amino acid residue. While some N‐terminal residues result in metabolically stable proteins, other, so‐called destabilizing residues, lead to rapid protein turnover. The N‐end rule pathway, which mediates the recognition and degradation of proteins with N‐terminal destabilizing residues, is present in all organisms examined, including prokaryotes. This protein degradation pathway has a hierarchical organization in which some N‐terminal residues, called primary destabilizing residues, are directly recognized by specific ubiquitin ligases. Other destabilizing residues, termed secondary and tertiary destabilizing residues, require modifications before the corresponding proteins can be targeted for degradation by ubiquitin ligases. In eukaryotes, the N‐end rule pathway is a part of the ubiquitin/proteasome system and is known to play essential roles in a broad range of biological processes in fungi, animals and plants. While the structure of the N‐end rule pathway has been extensively studied in yeast and mammals, knowledge of its organization in plants is limited. Using both tobacco and Arabidopsis, we identified the complete sets destabilizing and stabilizing N‐terminal residues. We also characterized the hierarchical organization of the plant N‐end rule by identifying and determining the specificity of two distinct N‐terminal amidohydrolases (Nt‐amidases) of Arabidopsis that are essential for the destabilizing activity of the tertiary destabilizing residues Asn and Gln. Our results indicate that both the N‐end rule itself and mechanistic aspects of the N‐end rule pathway in angiosperms are very similar to those of mammals.  相似文献   

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