Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine. Allylglycine-containing ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest. A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites. The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (K(i)s) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84(A)Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site. Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84(A)Gly. This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent.
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PMID:Chemically mediated site-specific proteolysis. Alteration of protein-protein interaction. 1185 28

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [(2)H(3)]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M(+1) and M(+2) mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3-10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.
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PMID:Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry. 1238 55

Vigilin, a member of the KH protein family, is exceptional among these proteins as it contains 14 KH domains in consecutive order. Vigilin is present in the nucleus and the cytoplasm of all eucaryotic cells studied so far and has apparently high affinity to tRNA and mRNA. There is circumstantial evidence that vigilin expression parallels high translational activity as demonstrated for pancreatic cells in vitro and in vivo as well as for carcinoma cell lines. On a molecular level we have recently demonstrated that vigilin promotes in vitro the export of tRNA from the nucleus to the translational machinery in the cytoplasm and may hence function as an intercompartimental conveyor. In the present study we show that exposure to a vigilin antisense oligo DNA (VAOD) expectedly resulted in a decrease of vigilin-expression, and was concomitant to lower amylase- and trypsin synthesis in freshly isolated pancreatic cells. In addition, carcinoma cells reacted with an increased mortality under exposure to VAOD giving further support for the notion that vigilin participates in cellular life-sustaining processes such as protein translation.
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PMID:Protein synthesis of eucaryotic cells could be decreased by antisense-DNA of the multi KH domain protein vigilin. 1279 6

The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.
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PMID:Changes in the extracellular proteome caused by the absence of the bldA gene product, a developmentally significant tRNA, reveal a new target for the pleiotropic regulator AdpA in Streptomyces coelicolor. 1583 21

The tRNA:m2(2)G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)--containing N-terminal domain [1-152] and C-terminal catalytic domain [157-329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPalpha) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPalpha and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPalpha structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNA(Asp) substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.
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PMID:THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain. 1668 54

The fusA gene encoding a thermophilic protein EF-G with multiple rare condons was cloned from Thermoanaerobacter tengcongensis (TteEF-G) and overexpressed in Escherichia coli by cotransfering a RIG plasmid to overcome the potential codon-bias problem originated from Arg, Ile and Gly. The recombinant protein was identified by MALDI-TOF-MS for molecular mass with approximation of 76 kDa and by trypsin digestion coupled LC-MS/MS for peptide sequence coverage of 61.3%. The in vivo complementary assay indicates that TteEF-G could significantly rescue the E. coli LJ14 (frr(ts)) at the non-permission temperature of 42 degrees C in the bi-transformant of TteRRF and TteEF-G. This study indicated that coexpression of rare codons' cognate tRNA is a useful method for protein overexpression in E. coli.
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PMID:Overexpression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis elongation factor G with multiple rare codons. 1797 23

Tetracycline (Tc) is a broad-spectrum antibiotic that kills bacteria by interrupting protein biosynthesis. It is thought that the bacteriostatic action of Tc is associated with its binding to the acceptor site (or A site) in the bacterial ribosome, interfering with the attachment of aminoacyl-tRNA. Recently, however, the crystal structure of a complex between Tc and trypsin-modified elongation factor Tu (tm-EF-Tu) was determined, raising the question of whether Tc binding to EF-Tu has a role in its inhibition of protein synthesis. We address this question using computer simulations. As controls, we first compute relative ribosome binding free energies for seven Tc variants for which experimental data are available, obtaining good agreement. We then consider the binding of Tc to both the trypsin-modified and unmodified EF-Tu-GDP complexes. We show that the direct contribution of EF-Tu to the binding free energy is negligible; rather, the binding can be solely attributed to interactions of Tc with a bridging Mg(2+) ion and the GDP phosphate groups. The effects of trypsin modification are modest. Further, our calculations show that EF-Tu does not exhibit any binding preference for Tc over the nonantibiotic, 4-dedimethyl-Tc, and EF-Tu does not bind the Tc analogue tigecycline, which is a potent antibiotic. In contrast, both the ribosome and the Tet Repressor protein (involved in Tc resistance) do show a binding preference for Tc over 4-dedimethyl-Tc, and the ribosome prefers to bind tigecycline over Tc. Overall, our results provide insights into the binding properties of tetracyclines and support the idea that EF-Tu is not their primary target.
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PMID:Binding of tetracyclines to elongation factor Tu, the Tet repressor, and the ribosome: a molecular dynamics simulation study. 1903 78

Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and l-cysteine to form l-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-O-[N-(l-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 A. The refined structure exhibited electron density corresponding to the entire 47 kDa MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions.
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PMID:The 1.6 A crystal structure of Mycobacterium smegmatis MshC: the penultimate enzyme in the mycothiol biosynthetic pathway. 1905 70

We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA-mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by approximately 20 A), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G*GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.
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PMID:Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome. 1953 29

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNA(Ile), the 2',3'-dialdehyde derivative of tRNA(Ile) was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNA(Ile) aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNA(Ile)ox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNA(Ile) and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence (601)KMSKS(605). Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [(32)P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.
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PMID:Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry. 1955 55


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