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)

Here, we report on the facilitated reactivation (85%) of oxidatively inactivated rhodanese by an oxidized form of the molecular chaperone GroEL (ox-GroEL). Reactivation by ox-GroEL required a reductant, and the enzyme substrate, sodium thiosulfate. Also, we found that ox-GroEL formed a complex with oxidatively inactivated rhodanese as shown by differential centrifugation and fluorescence spectroscopy. Ox-GroEL was obtained upon incubation of native GroEL for 16 h with 5 mM hydrogen peroxide. Under these conditions, GroEL was shown to retain its quaternary and secondary structures, but it displayed an increased exposure of hydrophobic surfaces as detected with 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence. Additionally, ox-GroEL was significantly more sensitive towards proteolysis with trypsin compared to the native form of the protein. The oxidatively inactivated form of rhodanese, also had an increased exposure of hydrophobic surfaces, as previously reported. Thus, the proteins binding appeared to be mediated by hydrophobic interactions. Unlike in prior reactivation studies that involved native GroEL or alpha-crystallin, we have clearly shown that an oxidized form of GroEL can function as a molecular chaperone in the reactivation of oxidatively inactivated rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.
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PMID:Oxidized GroEL can function as a chaperonin. 1476 3

Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, we investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the eluant. Upon immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known to be a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury.
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PMID:73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin. 1496 37

Mutation of all nonessential cysteine residues to serines in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). bis-ANS binding studies have shown that C3S has more hydrophobic exposure than WT, although both have similar secondary structures suggesting the flexibility of its structure. Activity of C3S falls once it binds bis-ANS, and covalent binding of bis-ANS to C3S is induced by light. bis-ANS binds to C3S in its C-terminal domain as is shown by gel electophoresis and proteolysis. bis-ANS binding makes the C-terminal domain more susceptible to trypsin cleavage.
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PMID:Active rhodanese lacking nonessential sulfhydryl groups has increased hydrophobic exposure not observed in wild-type enzyme. 1521 96

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, ¹H-¹⁵N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.
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PMID:The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients. 3265 Dec 34


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