Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Dendritic cells from the mesenteric lymph nodes (MLN) contain dense esterase-positive inclusions that may originate in effete intestinal epithelial cells and reach MLN without degradation. The MLN esterases have the electrophoretic mobilities of both intestinal and mononuclear cells. Cryptosporidium parvum (CP)-infected mice have CP Ag-positive cells in MLN and also increased numbers of dense esterase-positive cells, but the CP Ag-positive cells do not stain for esterase. To characterize the handling of epithelial cell products by dendritic cells, we analyzed mRNAs in the MLN of control and CP-infected recombination-activating gene(-/-)DO11.10 mice by oligoarrays. mRNAs for 115 proteins were increased in MLN after CP infection, of which the principal increases in trypsin and chymotrypsin approximated to 250-fold. Colipase, reg-1, C-reactive protein-ductin, and amyloid were also up-regulated >10-fold and all returned to baseline by 28 days after infection. mRNAs for the same proteins were detected in intestinal epithelial cells of infected mice by oligoarrays and RT-PCR after infection. mRNA for CP beta-tubulin was detectable in intestinal epithelial cells between 5 and 18 days after infection but was not detected in the MLN throughout the observation period. It appears that host response to CP infection includes expression of mRNA for some pancreatic enzymes by intestinal epithelial cells and their subsequent transport to the MLN. The esterase and trypsin, and mRNAs for chymotrypsin, colipase, and others that may derive from uninfected epithelial cells, appear to be transported to the MLN intact, while mRNA for CP beta-tubulin that is derived from infected cells is degraded.
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PMID:Intact intestinal mRNAs and intestinal epithelial cell esterase, but not Cryptosporidium parvum, reach mesenteric lymph nodes of infected mice. 1167 48

Cryptophycin-1 is the parent compound of a group of cyclic peptides with potent antineoplastic activity. Cryptophycins are thought to function by modulating the dynamic instability of spindle microtubules, and in vitro are known to bind in an equimolar ratio to the beta-tubulin subunit and to induce the formation of ring-like complexes. However, the detailed mechanisms whereby the cryptophycins interact with tubulin are not known. We have investigated the origin of the conformational changes in tubulin both biochemically and by electron microscopy and image analysis. Cryptophycin was found to protect both alpha- and beta-tubulin against proteolysis by trypsin, indicating conformational changes in specific regions of both subunits. The ring mass was determined to be approximately 0.81 MDa by sedimentation velocity combined with dynamic light scattering and by STEM, indicating a complex of eight alphabeta dimers. Statistical analysis of rings imaged by cryoelectron microscopy revealed 16-fold symmetry, corresponding to eight dimers. Computational averaging based on this symmetry yielded an image of a 24 nm diameter ring, at 2.6 nm resolution, that clearly distinguishes intradimer contacts from interdimer contacts, and allows discrimination of alpha-subunits from beta-subunits. Fitting of the tubulin dimer crystal structure into this projected density map indicates two points of curvature: a 13 degrees intradimer bend and a 32 degrees interdimer bend. We conclude that drug binding to one subunit (beta) results in two bends per dimer, affecting both subunits.
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PMID:The cryptophycin-tubulin ring structure indicates two points of curvature in the tubulin dimer. 1237 8

Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.
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PMID:Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry. 1537 91

We have shown that beta-tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl-N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tert-butyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60:985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[125I]CEU was effectively bound to the modified beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of beta-tubulin confirmed binding of iodophenylethylureido moiety to peptides [175-213] or [197-208] respectively. Fragmentation analyses by electrospray mass spectrometry using triply charged ions of peptide [175-213] identified a glutamic acid at position 198 as target for alkylation via an ester bond with ICEU. This amino acid located in the intermediate domain of the beta-tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament.
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PMID:Alkylation of beta-tubulin on Glu 198 by a microtubule disrupter. 1609 45

Rat primary microglia (MG) acquired a multipotent property to give rise to neuroectodermal cells through two-step culture in 10 and 70% serum-supplemented media for 5 days. Such multipotent MG, called promicroglioblasts (ProMGBs), formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2+ MG, which appeared to be the source of NG2+ ProMGB aggregates. The aggregates were MG marker+/NG2+/GFAP+/NCAM+/S-100beta- and had alkaline phosphatase activity. The marked accumulation of NG2+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2+ MG in the wounds separated with trypsin-EDTA formed NG2+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing beta-tubulin III+ cells in vitro easily. These data suggest that NG2+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain.
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PMID:NG2 proteoglycan-expressing microglia as multipotent neural progenitors in normal and pathologic brains. 1653 76

Abnormalities of the anterior cingulate cortex have previously been described in schizophrenia, major depressive disorder and bipolar disorder. In this study 2-DE was performed followed by mass spectrometric sequencing to identify disease-specific protein changes within the anterior cingulate cortex in these psychiatric disorders. The 2-DE system comprised IPGs 4-7 and 6-9 in the first, IEF dimension and SDS-PAGE in the second dimension. Resultant protein spots were compared between control and disease groups. Statistical analysis indicated that 35 spots were differentially expressed in one or more groups. Proteins comprising 26 of these spots were identified by mass spectroscopy. These represented 19 distinct proteins; aconitate hydratase, malate dehydrogenase, fructose bisphosphate aldolase A, ATP synthase, succinyl CoA ketoacid transferase, carbonic anhydrase, alpha- and beta-tubulin, dihydropyrimidinase-related protein-1 and -2, neuronal protein 25, trypsin precursor, glutamate dehydrogenase, glutamine synthetase, sorcin, vacuolar ATPase, creatine kinase, albumin and guanine nucleotide binding protein beta subunit. All but three of these proteins have previously been associated with the major psychiatric disorders. These findings provide support for the view that cytoskeletal and mitochondrial dysfunction are important components of the neuropathology of the major psychiatric disorders.
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PMID:Proteomic analysis of the anterior cingulate cortex in the major psychiatric disorders: Evidence for disease-associated changes. 1663 10

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in the nucleus accumbens (NAc), a brain region associated with drug reinforcement. Two-dimensional gel electrophoresis was used to compare protein alterations in the NAc between cocaine overdose (COD) victims (n=10) and controls (n=10). Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight. A total of 1407 spots were found to be present in a minimum of five subjects per group and the intensity of 18 spots was found to be differentially abundant between the groups, leading to positive identification of 15 proteins by peptide mass fingerprinting (PMF). Of an additional 37 protein spots that were constitutively expressed, 32 proteins were positively identified by PMF. Increased proteins in COD included beta-tubulin, liprin-alpha3 and neuronal enolase, whereas decreased proteins included parvalbumin, ATP synthase beta-chain and peroxiredoxin 2. The present data provide a preliminary protein profile of COD, suggesting the involvement of novel proteins and pathways in the expression of this complex disease. Additional studies are warranted to further characterize alterations in the differentially regulated proteins. Understanding the coordinated involvement of multiple proteins in cocaine abuse provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention for drug abuse-related disorders.
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PMID:Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims. 1707 5

Class III beta-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function.
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PMID:Proteomic characterization of cytoskeletal and mitochondrial class III beta-tubulin. 1864 17

Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide- and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein beta-tubulin after two distinct Arg residues. Neither GrK cleavage sites in beta-tubulin nor a peptide-based proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.
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PMID:Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A. 1905 12

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as "built-in" internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from betaI-, betaII-, betaIII-, betaIVb-, and betaV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the beta-tubulin isotypes. To show the utility of this method, we determined that betaI-tubulin represented 77% and betaIII-tubulin represented 3.2% of the total HeLa beta-tubulin.
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PMID:A label-free mass spectrometry method for the quantification of protein isotypes. 1968 26


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