Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg⁹⁸ and Arg¹⁰¹ residues, whereas plasmin only cleaved the propeptide downstream of Arg¹⁰¹. Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn¹⁰⁹-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.Matrix metalloprotease (MMP)³-2 is a member of the zinc-dependent endopeptidase family, which comprises 24 enzymes (1). MMP-2 plays a key role in many biological and pathological processes, including organ growth, endometrial cycling, wound healing, bone remodeling, tumor invasion, and metastasis (2). This enzyme functions through proteolysis of non-structural extracellular molecules and components of the basement membrane, including type IV collagen, fibronectin, elastin, laminin, aggrecan, and fibrillin (3).Like most MMPs, MMP-2 is synthesized as a zymogen that is activated by conformational change (4) or proteolysis within the propeptide, which may involve membrane type MMPs (MT-MMPs) (5–9). The most studied activation mechanism for pro-MMP-2 is cleavage of the propeptide by MT1-MMP, which requires cooperative activity between MT1-MMP and tissue inhibitor of metalloprotease (TIMP)-2 (5, 10–12). Serine proteases, such as thrombin, factor Xa, activated protein C, and plasmin as well as the cysteine protease legumain are all known activators of pro-MMP-2 (13–17).In addition to its role in coagulation, thrombin is involved in multiple cellular processes, including mitogenesis of fibroblasts (18), lymphocytes (19), mesenchymal cells (20), and smooth muscle cells (SMCs) (21, 22). Factor Xa acts as a potent mitogen for endothelial cells (23), fibroblasts (24), and vascular SMCs (25, 26). Both proteases can also elicit endothelial cell and SMC migration through pro-MMP-2 activation and subsequent extracellular matrix degradation (13, 27, 28). However, despite studies suggesting that MT1-MMP is involved in thrombin-mediated activation of pro-MMP-2, a detailed mechanism for MMP-2 activation has yet to be elucidated (15, 27).In this study, we investigated the roles of factor Xa and thrombin in MMP-2 regulation. Data are presented to demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 by cleavage of specific sites within the propeptide. Furthermore, factor Xa-processed MMP-2 showed enzymatic activity that was enhanced following intermolecular autoproteolytic cleavage. Thrombin also activated pro-MMP-2 through the same cleavage reaction. Interestingly, factor Xa and thrombin were also found to be involved in MMP-2 degradation. However, this activity was reduced greatly in thrombin-treated MMP-2 by the cell surface, which resulted in an increase in processed MMP-2.     

Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.Angiogenesis, the formation of blood vessels from pre-existing blood vessels, is a complex process essential for repairing injured tissue or supporting tissue growth. A great deal of work has been done to focus on understanding this phenomenon as it occurs in vivo, in particular with regard to its roles in embryonic development (1–5). In contrast to embryonic development, adult angiogenesis and inflammation are closely related phenomena that occur in vivo in a number of physiologically relevant processes. Inflammation lies at the crux of multiple physiological events in biological systems that precede the induction of angiogenesis: wound healing (6–8), chronic wounds (8), inflammatory disorders (9, 10), and cancer (9, 11, 12).Inflammatory reactions also confound tissue engineered implantable three-dimensional constructs that provide innovative clinical treatments of various diseases and injuries (13–17). As complex tissues become developed for applications in clinical trials, tissue vascularization for constructs of considerable size and volume is required for their survival (18, 19). Once implanted, these constructs will also experience significant inflammatory responses within their host''s local milieu (20, 21). These circumstances demonstrate the necessity for understanding the interactions between inflammation and angiogenesis, such as the development of predictive models (22). Elucidating specific intracellular mechanisms can provide insight for novel approaches in treatment of diseases as well as predicting responses to artificially engineered tissues.Recently, studies have shown that chemokines, which play a central role in inflammation, can influence the outcomes of angiogenesis (23–26) by promoting new blood vessel growth (e.g. CXCL1–3, CXCL5–8, CXCL12) or inhibiting its formation altogether (e.g. CXCL4, CXCL9–11, CXCL13) (26). In particular, a large body of information is available on platelet factor 4 (PF-4/CXCL4) and its ability to inhibit and even induce regression of angiogenesis. PF-4 is found throughout the adult body, at roughly 0.25–1.25 nm (2–10 ng/ml) in blood plasma, but as high as 25 μm in localized areas during wound healing (27, 28). Its ubiquitous presence, implication in cancer and vascular diseases, and use as a potential drug therapy have made PF-4 a key point of interest in influencing angiogenesis in vivo (27–30). In addition to inducing angiostasis, PF-4 can inhibit cell proliferation by halting S phase progression and reducing endothelial cell migration (25, 28, 30–32). Despite the wealth of information on PF-4 and its mechanistic effects on immune cells, scarce literature exists on the nature of the molecular signaling with endothelial cells to inhibit angiogenesis. Furthermore, the complexity of PF-4 mediated signaling and its potential to interact through multiple binding mechanisms makes it difficult to determine how PF-4 can interfere with angiogenesis (28, 29, 33, 34). Possible angiogenic signaling network interference mechanisms for PF-4 include the sequestration of growth factors and proteoglycans, antagonism of integrin-mediated signaling, or direct signaling through its chemokine receptor CXCR3, all of which have supporting evidence in previous literature (28). Along with the multiple mechanisms PF-4 may utilize for signaling, only limited studies on direct signaling elicited by PF-4 on endothelial cells have been reported; one of interest found that P38 MAPK can be activated via CXCR3 on endothelial cells cultured on plastic (35), whereas another, more definitive study showed PF-4 acting similarly to other CXCR3 ligands in activating PKA to prevent m-calpain-mediated rear de-adhesion of moving cells (36, 37). Furthermore, PF-4 could have variable sensitivities in different endothelial cell types because of heterogeneous expression of CXCR3 (38).In our study, we sought to develop an approach to assess network-level signaling interactions between PF-4 and the major angiogenic inducer vascular endothelial growth factor (VEGF)¹ within a contextually relevant 3-D angiogenesis platform, in a controlled environment to understand what role these two factors may play. We developed methods to reduce extracellular matrix contamination in our samples and were able to successfully use a two-step lysis method with a MS compatible detergent-based lysis buffer. By taking advantage of iTRAQ-based multiplexed quantitation, we were able to collect quantitative phosphoprotein signaling data from our system with early and late temporal resolution. Using correlation network methods to observe differences in our system, we found that simultaneous treatment with PF-4 and VEGF induced changes in migrational pathway topology when compared with VEGF treatment alone. Most often, these changes appeared as losses in correlations between different migrational signaling proteins. We found that several different signaling pathways involved with migration were affected, including central proteins P38α MAPK, focal adhesion kinase (FAK), and Src family kinases. Furthermore, we found statistically significant differences in tyrosine phosphorylation when HDMVECs were stimulated with VEGF and PF-4, as opposed to only VEGF. In addition, we were able to recapitulate previously reported findings on how PF-4 infers its angiostatic effects on endothelial cells. Surprisingly, our data set revealed EphA2 receptor as a central node for PF-4 signaling, indicating that it may possess a complementary role in the balance of angiogenic and angiostatic effects.To our knowledge, this is the first attempt at performing MS-based analysis of phosphotyrosine signaling networks within the context of an environment that is amenable to angiogenesis. Our work provides a step forward in applying high throughput and systems-level phosphoproteomics data collection to more physiologically relevant experimental conditions.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Commitment and Effector Phases of the Physiological Cell Death Pathway Elucidated with Respect to Bcl-2, Caspase,and Cyclin-Dependent Kinase Activities

Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1β-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.Although interest in the process of physiological cell death has grown enormously in recent years, the mechanism of death has remained enigmatic. While the induction of physiological death in diverse cell types is effected by a wide variety of stimuli, a common morphology, described as apoptosis, ensues in all cases. The commonality of morphology has led to the belief that disparate inducers trigger distinct signaling events which ultimately converge in a common biochemical pathway of death. This hypothesis suggests a division of the biochemical process into upstream events that are specific for individual inducers and downstream steps, comprising the common pathway, which bring about the actual demise of the cell.Since most cell deaths in the nematode Caenorhabditis elegans are induced in a lineage-determined program, the simple pathway of death elucidated in that species (17) is likely to be revealing of downstream steps. Cell death in C. elegans is dependent on the activation of Ced3, a cysteine protease (77, 79), and is inhibited by Ced9 (27). In mammalian cells, a group of Ced3 homologs, termed caspases (1), appears to play a role in virtually all of the physiological cell deaths studied to date. These enzymes cleave on the carboxyl-terminal side of aspartate residues within distinct recognition motifs. Each caspase is synthesized as a proenzyme and activated by cleavage at internal sites, potentially by the same or another caspase class (66, 77). This leads to the notion that caspases function in an ordered cascade, with members of one family activating members of the next. Data consistent with this pattern have been obtained from studies in vitro (41, 60, 65).Of the large family of mammalian caspases, caspase 3 is closely homologous to Ced3 and appears to be involved widely in cell deaths (50, 65). Nonetheless, specific caspases seem not to be associated uniquely with distinct cases of death, and gene-targeting experiments reveal that the absence of a single caspase has extremely limited consequences for cell death responsiveness (38, 39).Similarly, a family of ced9-related death response modulatory genes exists in mammals; the most closely related homolog, bcl-2, is functionally interchangeable with ced9 in the worm (28, 73). These gene products do not function in all mammalian cell deaths (61, 72). Moreover, while the products of some bcl-2 gene family members have death-sparing activity (6, 7), others exert the opposite effect (52, 78).Several cellular proteins, among them poly(ADP-ribose) polymerase (PARP), nuclear lamins, fodrin, and DNA-dependent protein kinase (10, 16, 34), are targets for cleavage by various caspases. In cells spared from death, for example by Bcl-2, these proteolytic events do not occur (9, 13, 18). Still, the cleavage of none of these proteins has been shown to be essential for the cell death response (42, 54, 74). The specific consequences of caspase activation which are lethal are unknown.It may be that the consequence of protease activity is the specific activation of distinct death effectors. We have proposed that essential genes involved in cell division may be critically involved in cell death as well and that the difficulty in identifying distal effector steps genetically reflects the indispensable function of those gene products in cell life (67). Data from several groups have shown that cell cycle catastrophes, the precocious expression of mitosis-like cyclin-dependent histone kinases (Cdks), are associated with a variety of physiological cell deaths and that the inhibition of death by Bcl-2 is associated with alterations in the expression and localization of these Cdk proteins (22, 23, 29, 36, 40, 46, 47, 58, 59, 70).We have taken advantage of the death-sparing activities of Bcl-2 and two viral caspase inhibitors, CrmA and p35 (64, 77), to dissect the mechanism of cell death in two separate cellular paradigms. These studies allow us to draw a generalized skeletal pathway of the death-associated biochemical activities discussed above and demonstrate the requisite involvement of these different classes of activities in a conserved and ordered pathway by which cells die physiologically.     

Secreted Versus Membrane-anchored Collagenases: RELATIVE ROLES IN FIBROBLAST-DEPENDENT COLLAGENOLYSIS AND INVASION*

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13^−/−, Mmp8^−/−, Mmp2^−/−, Mmp9^−/−, Mmp14^−/− and Mmp16^−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14^−/− or Mmp16^−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14^−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.In the postnatal state, fibroblasts are normally embedded in a self-generated three-dimensional connective tissue matrix composed largely of type I collagen, the major extracellular protein found in mammals (1–3). Type I collagen not only acts as a structural scaffolding for the associated mesenchymal cell populations but also regulates gene expression and cell function through its interactions with collagen binding integrins and discoidin receptors (2, 4). Consistent with the central role that type I collagen plays in defining the structure and function of the extracellular matrix, the triple-helical molecule is resistant to almost all forms of proteolytic attack and can display a decades-long half-life in vivo (4–6). Nonetheless, fibroblasts actively remodel type I collagen during wound healing, inflammation, or neoplastic states (2, 7–13).To date type I collagenolytic activity is largely confined to a small subset of fewer than 10 proteases belonging to either the cysteine proteinase or matrix metalloproteinase (MMP)² gene families (4, 14–18). As all collagenases are synthesized as inactive zymogens, complex proteolytic cascades involving serine, cysteine, metallo, and aspartyl proteinases have also been linked to collagen turnover by virtue of their ability to mediate the processing of the pro-collagenases to their active forms (13, 15, 19). After activation, each collagenase can then cleave native collagen within its triple-helical domain, thus precipitating the unwinding or “melting” of the resulting collagen fragments at physiologic temperatures (4, 15). In turn, the denatured products (termed gelatin) are susceptible to further proteolysis by a broader class of “gelatinases” (4, 15). Collagen fragments are then either internalized after binding to specific receptors on the cell surface or degraded to smaller peptides with potent biological activity (20–24).Previous studies by our group as well as others have identified MMPs as the primary effectors of fibroblast-mediated collagenolysis (20, 25, 26). Interestingly, adult mouse fibroblasts express at least six MMPs that can potentially degrade type I collagen, raising the possibility of multiple compensatory networks that are designed to preserve collagenolytic activity (25). Four of these collagenases belong to the family of secreted MMPs, i.e. MMP-13, MMP-8, MMP-2, and MMP-9, whereas the other two enzymes are members of the membrane-type MMP subgroup, i.e. MMP-14 (MT1-MMP) and MMP-16 (MT3-MMP) (13, 27–29). From a functional perspective, the specific roles that can be assigned to secreted versus membrane-anchored collagenases remain undefined. As such, fibroblasts were isolated from either wild-type mice or mice harboring loss-of-function deletions in each of the major secreted and membrane-anchored collagenolytic genes, and the ability of the cells to degrade type I collagen was assessed. Herein, we demonstrate that fibroblasts mobilize either secreted or membrane-anchored MMPs to effectively degrade type I collagen in qualitatively and quantitatively distinct fashions. However, under conditions where fibroblasts use either secreted and membrane-anchored MMPs to exert quantitatively equivalent collagenolytic activity, only MT1-MMP plays a required role in supporting a collagen-invasive phenotype. These data establish a new paradigm wherein secreted collagenases are functionally limited to bulk collagenolytic processes, whereas MT1-MMP uniquely arms the fibroblast with a focalized degradative activity that mediates subjacent collagenolysis as well as invasion.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Angioinhibitory Action of NK4 Involves Impaired Extracellular Assembly of Fibronectin Mediated by Perlecan-NK4 Association

NK4, a fragment of hepatocyte growth factor (HGF), exerts bifunctional action as a competitive antagonist against HGF and its receptor c-Met and an angiogenesis inhibitor. Here we studied the anti-angiogenic mechanism of NK4. In cultured human endothelial cells, NK4 inhibited DNA synthesis induced not only by HGF but also by either basic fibroblast growth factor or vascular endothelial growth factor. Even if c-Met expression was diminished by small interference RNA, NK4 inhibited basic fibroblast growth factor-induced DNA synthesis, indicating that anti-angiogenic action of NK4 is c-Met-independent. Affinity purification with NK4-immobilized beads revealed that NK4 binds to perlecan. Consistent with this, NK4 colocalized with perlecan in endothelial cells. Perlecan is a multidomain heparan sulfate proteoglycan that interacts with basement membrane components such as fibronectin. NK4 inhibited extracellular assembly of fibronectin, by which fibronectin-dependent endothelial cell spreading was inhibited by NK4. Knockdown of perlecan expression by small interference RNA significantly abrogated the inhibitory effect of NK4 on fibronectin assembly and cell spreading. In NK4-treated endothelial cells, tyrosine phosphorylation of focal adhesion kinase and Rac activation were reduced, whereas overexpression of activated Rac recovered the DNA synthesis in NK4-treated endothelial cells. These results indicate that the association between NK4 and perlecan impairs fibronectin assembly, thereby inhibiting anchorage-dependent signaling. The identified mechanism for angiostatic action provides further proof of significance for NK4 in the treatment of cancer and potentially for vascular regulation as well.The manipulation of angiogenesis has potential therapeutic value for the treatment of a variety of diseases including cancer, arthritis, and cardiovascular disease (1, 2). In addition to endothelial cell migration and proliferation, angiogenesis is a process involving dynamic matrix transition (3). During angiogenesis, the vascular basement membrane undergoes proteolytic degradation and transit to the provisional matrix consisting of fibronectin, etc., followed by an intermediate and mature new vascular basement membrane. Growing evidence has shown that such an extracellular matrix (ECM)² not only provides mechanical support to the cells but also essentially regulates cell growth, migration, and survival. The fact that a number of endogenous inhibitors of angiogenesis have been identified from proteolytic fragments of ECM molecules also highlights the important regulatory roles of ECM in angiogenesis (3).NK4 is a proteolytic fragment of hepatocyte growth factor, HGF (4), consisting of an N-terminal hairpin domain and four kringle domains of the α-chain of HGF (5). By competitively binding to HGF receptor c-Met, NK4 acts as an HGF antagonist (5, 6). The NK4 fragment seems to be physiologically generated by mast cells and neutrophils peptidases during inflammation (7). Because HGF regulates malignant behavior in a variety of tumors by inducing invasive, angiogenic, and metastatic responses (8, 9), the blockade of HGF-c-Met signaling by NK4 is a strategy to inhibit tumor invasion and metastasis (6, 9–11). During investigation of a therapeutic approach with NK4 in experimental cancer models, we unexpectedly found that NK4 functions as an angiogenesis inhibitor (12). Based on the bifunctional characteristic as HGF antagonist and angiogenesis inhibitor, NK4 suppressed malignant behavior of cancers, including invasion, metastasis, and angiogenesis-dependent tumor growth (9–12).The angiostatic activity of NK4 is probably independent of its original activity as an HGF antagonist because an anti-HGF antibody capable of preventing HGF-c-Met association did not inhibit human endothelial cell growth stimulated by either bFGF or VEGF (12). However, the mechanism by which NK4 inhibits angiogenic responses in endothelial cells remains to be addressed. In the present study we newly identified perlecan to be an NK4 binding molecule and found that in vascular endothelial cells the association of NK4 with perlecan inhibited extracellular fibronectin assembly, fibronectin-dependent cell spreading, and the subsequent anchorage-dependent signals. Together with our finding that c-Met/HGF receptor is not required for the inhibition of DNA synthesis by NK4, we propose that the association of NK4 with perlecan plays a key role in angiogenesis inhibition by NK4.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.