From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two‐chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X‐ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha‐carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha‐carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy‐terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this “occluding loop' provide positively charged anchors for the C‐terminal carboxylate group of such polypeptide substrates. These structural features explain the well‐known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2‐side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo‐ and exopeptidase activity. The “occluding loop' does not allow cystatin‐like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.
A ~eries of new epoxysuccinyl peplides were devil|ned and syntl~©sized to develop a s~eifl¢ inhibitor of ¢alhep~in B, Of these compounds, N-(L. 3-tr(uu-ethoxyearbo)i~oloueyI.L.proline tcompound CA.0741 were lhe mosl polcnt and q~¢ctl~c inhibitors of eathepstn B in vitro, The carboxyl llroup of prolinc and the (:thyl ester llroup or n.propylamide lift, up in the oxiran¢ rtn[I were necessary, the ethyl ©~ter ilroup or the n.propylamtt|e ~roup bein8 particularly effective fur distinlluishlnl$ eatltepsin B from other cystcine prot¢inases such as cathepsins L and H, and ealpains, Epox~'~ucciayI peptide; Cathepsin B: Cys~einc proteinase; Specific inhibitor
To elucidate the mechanisms of microgravity‐induced muscle atrophy, we focused on fast‐type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16‐d spaceflight (STS‐90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase‐polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin‐conjugating enzyme in the muscles, compared with those of ground control rats. The levels of μ‐calpain, m‐calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail‐suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up‐regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E‐64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin‐dependent proteolytic pathway.
New derivatives of E-64 (compound CA-030 and CA-074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10,000-30,000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L: their initial Ki values for cathepsin B were about 2-5 nM, like that of E-64-c, whereas their initial Ki values for cathepsins H and L were about 40 200 microM. In in vivo conditions, such as intraperitoneal injection of compound CA-030 or CA-074 into rats, compound CA-074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA-030 does not show selectivity for cathepsin B, although both compounds CA-030 and CA-074 show complete selectivity for cathepsin B in vitro.
(20) and actinidin (21).The experimental details of the sequence analysis of cathepsins B and H will be published elsewhere. MATERIALS AND METHODSCrystalline rat liver cathepsin B was prepared as described (11). Cathepsin H was prepared by the method of Schwartz and Barrett (22). The intact chain and the two polypeptide chains of each enzyme, generated by proteolytic cleavage, were separated on a Sephacryl S-200 column after S-carboxymethylation of reduced disulfide bonds as reported (19). Trypsin treated with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK-trypsin) and Staphylococcus aureus V8 protease were obtained from Worthington and Miles, respectively. Gel filtration media were products of Pharmacia.Chemical cleavages by cyanogen bromide (Eastman) and by 2 -(2 -nitrophenylsulphenyl) -3-methyl -3'-bromoindolenine (BNPS-skatole) (Pierce) were carried out by methods of Gross and Witkop (23) (30,31).A search for a homologous sequence was carried out with a VAX/VMS computer by using a "protein sequence database" of the Atlas of Protein Sequence and Structure (version 4, April 30, 1982) obtained from the National Biomedical Research Abbreviations: TPCK-trypsin, trypsin treated with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone; BNPS-skatole, 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine. 3666The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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