Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The low molecular weight polypeptide required for energy-dependent proteolysis, ubiquitin, is rapidly inactivated by 100,000 X g supernatants of rabbit liver extracts. Ubiquitin inactivation results from limited proteolysis by an endogenous contaminating lysosomal thiol protease having trypsin-like specificity. Evidence for this includes a pH optimum of 5.0 for the first order constant of ubiquitin inactivation and observation that inactivation is inhibited by EDTA, o-phenanthroline, iodoacetamide, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethyl ketone, leupeptin, soybean trypsin inhibitor, and aprotinin. Metals stimulate but are not required for ubiquitin inactivation with the effect apparently mediated by a low molecular weight heat-labile component of crude extracts. When this heat-labile component is removed by gel exclusion chromatography a number of metals inhibit ubiquitin inactivation. In the presence of excess dithiothreitol, inhibition is relatively specific for Zn(II). Inhibition by Zn(II) is specifically overcome competitively by Cd(II) or by a concentration of ubiquitin in excess of Zn(II). The responsible cathepsin possesses a molecular mass of 35 kDa by gel exclusion chromatography and shows marked thermal lability at neutral pH but stability at acid pH. Proteolytic inactivation of ubiquitin results from limited cleavage of the carboxyl-terminal glycine dipeptide required for isopeptide bond formation and is supported by data on isoelectric point changes on subsequent digestion with carboxypeptidase B and by direct amino acid analysis. When the responsible cathepsin is inactivated, liver extracts display ATP,ubiquitin-dependent proteolysis that cannot be ascribed to contaminating erythrocytes. Thus the previous inability to demonstrate energy-dependent proteolysis in liver extracts is accounted for by the artifactual inactivation of ubiquitin.
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PMID:The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts. 298 63

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.
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PMID:Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system. 300 81

Long dynamics simulations were carried out on the B1 immunoglobulin-binding domain of streptococcal protein G (ProtG) and bovine pancreatic trypsin inhibitor (BPTI) using atomistic descriptions of the proteins and a continuum representation of solvent effects. To mimic frictional and random collision effects, Langevin dynamics (LD) were used. The main goal of the calculations was to explore the stability of tens-of-nanosecond trajectories as generated by this molecular mechanics approximation and to analyze in detail structural and dynamical properties. Conformational fluctuations, order parameters, cross correlation matrices, residue solvent accessibilities, pKa values of titratable groups, and hydrogen-bonding (HB) patterns were calculated from all of the trajectories and compared with available experimental data. The simulations comprised over 40 ns per trajectory for ProtG and over 30 ns per trajectory for BPTI. For comparison, explicit water molecular dynamics simulations (EW/MD) of 3 ns and 4 ns, respectively, were also carried out. Two continuum simulations were performed on each protein using the CHARMM program, one with the all-atom PAR22 representation of the protein force field (here referred to as PAR22/LD simulations) and the other with the modifications introduced by the recently developed CMAP potential (CMAP/LD simulations). The explicit solvent simulations were performed with PAR22 only. Solvent effects are described by a continuum model based on screened Coulomb potentials (SCP) reported earlier, i.e., the SCP-based implicit solvent model (SCP-ISM). For ProtG, both the PAR22/LD and the CMAP/LD 40-ns trajectories were stable, yielding C(alpha) root mean square deviations (RMSD) of about 1.0 and 0.8 A respectively along the entire simulation time, compared to 0.8 A for the EW/MD simulation. For BPTI, only the CMAP/LD trajectory was stable for the entire 30-ns simulation, with a C(alpha) RMSD of approximately 1.4 A, while the PAR22/LD trajectory became unstable early in the simulation, reaching a C(alpha) RMSD of about 2.7 A and remaining at this value until the end of the simulation; the C(alpha) RMSD of the EW/MD simulation was about 1.5 A. The source of the instabilities of the BPTI trajectories in the PAR22/LD simulations was explored by an analysis of the backbone torsion angles. To further validate the findings from this analysis of BPTI, a 35-ns SCP-ISM simulation of Ubiquitin (Ubq) was carried out. For this protein, the CMAP/LD simulation was stable for the entire simulation time (C(alpha) RMSD of approximately 1.0 A), while the PAR22/LD trajectory showed a trend similar to that in BPTI, reaching a C(alpha) RMSD of approximately 1.5 A at 7 ns. All the calculated properties were found to be in agreement with the corresponding experimental values, although local deviations were also observed. HB patterns were also well reproduced by all the continuum solvent simulations with the exception of solvent-exposed side chain-side chain (sc-sc) HB in ProtG, where several of the HB interactions observed in the crystal structure and in the EW/MD simulation were lost. The overall analysis reported in this work suggests that the combination of an atomistic representation of a protein with a CMAP/CHARMM force field and a continuum representation of solvent effects such as the SCP-ISM provides a good description of structural and dynamic properties obtained from long computer simulations. Although the SCP-ISM simulations (CMAP/LD) reported here were shown to be stable and the properties well reproduced, further refinement is needed to attain a level of accuracy suitable for more challenging biological applications, particularly the study of protein-protein interactions.
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PMID:Long dynamics simulations of proteins using atomistic force fields and a continuum representation of solvent effects: calculation of structural and dynamic properties. 1595 66