Matrix metalloproteases (MMPs) play many important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis and cancer. Traditionally, MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival, however their mode of action remains unknown. We demonstrated that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1 which promotes aggregation through PAR1. Unexpectedly, we found that MMP-1 cleaved PAR1 at a novel site which strongly activated Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 greatly curtailed thrombogenesis under arterial flow conditions and inhibited thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a new target for the prevention of arterial thrombosis.

Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies by mechanisms that are not understood 1,2 . The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-κB, blocks apoptosis and induces transformation [3][4][5][6] . This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-κB p65. We show that MUC1 interacts with the highmolecular-weight IκB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKβ and IKKγ. Interaction of MUC1 with both IKKβ and IKKγ is necessary for IKKβ activation, resulting in phosphorylation and degradation of IκBα. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKβ-IKKγ in response to TNFα stimulation. TNFα-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKβ is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKβ and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKβ-NF-κB p65 pathway.Nuclear localization of NF-κB p65 was studied in HCT116 colon cancer and HeLa cervical cancer cells that stably express either an empty vector or MUC1 (ref. 4, also see Supplementary Information, Fig. S1a). Levels of nuclear NF-κB p65 were lower in vector cells than in cells expressing MUC1 (Fig. 1a). Human ZR-75-1 and MCF-7 breast cancer cells that express endogenous MUC1 were stably transfected to express either an empty vector or a MUC1 siRNA 4 ( Supplementary Information, Fig. S1a). Silencing of MUC1 in ZR-75-1 (ref. 4) and MCF-7 cells 7 decreased nuclear NF-κB p65 (Fig. 1b). MUC1 expression was also associated with a decrease in cytosolic NF-κB p65 levels in HeLa and ZR-75-1 cells ( Supplementary Information, Fig. S1b). To determine whether MUC1 is associated with activation of the NF-κB p65 transcription function, HeLa and ZR-75-1 cells were transfected with a construct containing a NF-κB-binding site upstream of the luciferase reporter (pNF-κB-Luc). MUC1 expression was associated with activation of pNF-κB-Luc (Fig. 1c). In contrast, MUC1 had no effect on activation of a pNF-κB-Luc construct that was mutated at the NF-κB p65 binding site (Fig. 1c). In addition, expression of Bclx L , a gene activated by NF-κB, was higher in cells expressing MUC1 (Fig. 1d). To determine whether MUC1 affects IκBα phosphorylation (as phosphorylated IκBα is targeted for ubiquitination and proteosomal degradation) cytosolic lysates were immunoblotted with an anti-phospho-IκBα antibody. Indeed, phospho-IκBα levels were significantly higher in cells expressing MUC1 (Fig. 1e). Assessment of IκBα stability indicated that MUC1 expression increases degradation of IκBα (Fig. 1f). The half-lives of IκBα in the absence and presence of MUC1, were 6.7 ± 0.5 h and 3. Fig. 2a). In vitro studies with puri...

Serine hydroxymethyltransferase (SHMT), a member of the ␣-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.Serine hydroxymethyltransferase (SHMT; 1 EC 2.1.2.1) is a PLP-dependent enzyme that plays a central role in the onecarbon metabolism. It catalyzes the reversible inter-conversion of serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate, a key intermediate in the biosynthesis of purine, thymidine, choline, and methionine (1, 2). In addition to this physiological reaction, SHMT has also been shown to catalyze THF-independent aldolytic cleavage, decarboxylation, racemization, and transamination reactions (3). The importance of SHMT in DNA synthesis coupled with the observed high level of enzyme activity in rapidly proliferating cells has focused attention on SHMT as a potential target for the development of anticancer and antimicrobial agents (4 -6).Several mechanisms have been proposed for the hydroxymethyl transfer, the most favored being the retroaldol cleavage (7,8). The crystal structures of human liver SHMT (hcSHMT) and rabbit liver SHMT (rcSHMT) and Escherichia coli SHMT (eSHMT) as well as murine cytoplasmic SHMT (mcSHMT) have been reported (9 -12). The structure of a reduced form of rcSHMT representing a gem diamine equivalent has also been reported (10). Although these structures have provided a wealth of information regarding the architecture of the enzyme, active site, and residues involved in substrate binding and catalysis, several aspects of SHMT catalytic mechanism remain uncertain (7, 13). A detailed comparison and analysis of several structures of the enzyme corresponding to different intermediate steps and in complex with various substrates, substrate analogs, and product analogs are required to unravel the finer molecular details of ...

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The rapid and highly sensitive detection of extremely short-lived nitric oxide (NO) gas generated in vivo by a water-soluble fluorescein derivative is developed. This assay system comprises of indole-3-carboxaldehyde functionalized fluorescein hydrazone (FI) assay which displays a typically high absorption at 492 and 620 nm in the presence of Cu2+ and also shows FRET induced fluorescence turn-on exclusively with Cu2+. FI selectively detects Cu2+ in vivo and in vitro by the "turn-on" mechanism followed by fluorescence "turn-off" with NO gas generated by the lipopolysaccharide (LPS) action. The in vivo experiment performed in the cellular system indicates that FI loaded RAW264.7 cells showed bright fluorescence in the presence of Cu2+, while other metals did not influence the FI fluorescence. In addition, the fluorescence of FI-Cu2+ was efficiently quenched by NO generated in macrophages through LPS stimulation. FI demonstrates characteristic "turn-on" behavior in the presence of Cu2+ via spirolactom ring-opening, while other metals such as Na+, K+, Ca2+, Cr3+, Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+, and Ag+ did not influence FI fluorescence even at very high concentration. Further, the FI-Cu2+ complex fluorescence was not quenched with any anions or amino acids but totally quenched by NO and the paramagnetic nature of Cu2+ ion converted into the diamagnetic nature when reduced to Cu1+. FI and the FI-Cu2+ complex are nontoxic to the cellular system and have high potential for biomedical applications.

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