No abstract

Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. Elastin is an extracellular matrix protein composed of highly cross-linked, hydrophobic tropoelastin monomers which provides resilience to elastic fibers. The hydrophobicity and extensive cross-linking of tropoelastin monomers result in an insoluble elastic fiber which is highly resistant to proteolysis (1). Thus, under normal physiologic conditions, elastin undergoes minimal turnover (2). However, certain pathologic situations, including pulmonary emphysema (3) and abdominal aortic aneurysm (4), are characterized by proteolytic destruction of elastic fibers. The involvement of serine proteases in such pathologies has long been suspected. More recently, participation of cysteine proteinases (5) and matrix metalloproteinases (MMPs, 1 6 -8) in these diseases has been proposed. The MMPs comprise a gene family that collectively is capable of degrading all components of extracellular matrix in physiologic and pathologic states (9, 10). As presently recognized, this family consists of fibroblast (11), neutrophil (12), and breast carcinoma-derived (13) collagenases, three stromelysins, 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME, MMP-12), matrilysin, and a recently described 66-kDa membrane-type metalloproteinase (14). These enzymes are organized into homologous structural domains, with some differences in domain composition and number. All members share a zymogen domain, containing a conserved PRCGXPD motif involved in enzyme latency, and a zinc-binding CD. Most members also contain a hemopexin-like domain at their C terminus, the exception being matrilysin, which lacks this domain completely. Unique to the 72-kDa and 92-kDa gelatinases is an additional domain composed of three fibronectin type II-like repeats inserted in tandem within the zinc-binding CD. The 92-kDa gelatinase also contains an ␣2(V) collagen-like domain not found in any of the other family members.The issue of substrate specificity has received considerable attention recently in MMP biology. The determinants which confer substrate specificity to these enzymes appear to be localized within discrete structural domains. For example, the ability of the collagenases to degrade triple-helical collagen requires the presence of the C-terminal hemopexin-like domain (15)(16)(17). In contrast, the stromelysins degrade a variety of substrates in a manner which is independent of the C-terminal hemopexin-like dom...

Methionine aminopeptidase (MetAP) enzymes require a divalent metal ion such as Mn(II), Fe(II), Co(II), Ni(II), or Zn(II) for its removal of the N-terminal methionine from newly synthesized proteins, but it is not certain which of these ions is most important in vivo. Metalloform-selective MetAP inhibitors could be valuable for defining which metals are physiologically relevant for MetAP activation and could serve as leads for development of new therapeutic agents. We have screened a library of 43 736 small drug-like molecules against Escherichia coli MetAP and identified two groups of potent and highly metalloform-selective inhibitors of the Co(II)-form, and of the Mn(II)-form, of this enzyme. Compound 1 is 790-fold more selective for the Co(II)-form, while compound 4 is over 640-fold more potent toward the Mn(II)-form. The X-ray structure of a di-Mn(II) form of E. coli MetAP complexed with the Mn(II)-form-selective compound 4 was obtained, and it shows that the inhibitor interacts with both Mn(II) ions through the two oxygen atoms of its free carboxylate group. The preferential coordination of the hard (oxygen) donors to Mn(II) may contribute to its superb selectivity toward the Mn(II)-form.

Atg4 is required for cleaving Atg8, allowing it to be conjugated to phosphatidylethanolamine on phagophore membranes, a key step in autophagosome biogenesis. Deconjugation of Atg8 from autophagosomal membranes could be also a regulatory step in controlling autophagy. Therefore, the activity of Atg4 is important for autophagy and could be a target for therapeutic intervention. In this study, a sensitive and specific method to measure the activity of two Atg4 homologs in mammalian cells, Atg4A and Atg4B, was developed using a fluorescence resonance energy transfer (FRET)-based approach. Thus LC3B and GATE-16, two substrates that could be differentially cleaved by Atg4A and Atg4B, were fused with CFP and YFP at the N- and C-terminus, respectively, allowing FRET to occur. The FRET signals decreased in proportion to the Atg4-mediated cleavage, which separated the two fluorescent proteins. This method is highly efficient for measuring the enzymatic activity and kinetics of Atg4A and Atg4B under in vitro conditions. Applications of the assay indicated that the activity of Atg4B was dependent on its catalytic cysteine and expression level, but showed little changes under several common autophagy conditions. In addition, the assays displayed excellent performance in high throughput format and are suitable for screening and analysis of potential modulators. In summary, the FRET-based assay is simple and easy to use, is sensitive and specific, and is suitable for both routine measurement of Atg4 activity and high-throughput screening.