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]     

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining demonstrated expression of β-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ∼95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises <1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8–10).Dental enamel formation is divided into secretory, transition, and maturation stages (11, 12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides (3, 13, 14). By the end of the secretory stage the enamel crystals are full-length and the enamel layer as a whole is as thick as it will ever be, but it has only about 14% of the mineral as it will have when the tooth erupts (15). Following the secretory stage there is then a transition during which the ameloblasts greatly reduce their secretion of enamel proteins (16) and convert to maturation ameloblasts (17). During the maturation stage, mineral is deposited exclusively on the sides of pre-existing enamel crystallites (18), which grow in width and thickness until further growth is prevented by contact with adjacent crystals (19, 20). During early maturation the percentage protein by weight drops from 30 to 2% (5), and half of the total enamel mineral is deposited. The final 30–35% of mineral is deposited in the absence of significant protein and allows the crystals to grow firmly against one another and to mechanically interlock (15).The major secretory stage enamel proteins are amelogenin (21, 22), ameloblastin (23–25), and enamelin (26, 27). These proteins function specifically during enamel formation, and the disease phenotypes exhibited by mice lacking these genes are confined to the developing teeth and include enamel agenesis (28–30). These genes are often deleted or are reduced to pseudogenes in vertebrates such as birds or baleen whales that evolved alternatives to developing teeth (31, 32). Although the enamel extracellular matrix proteins are critical for growing enamel crystals, they are not part of the final enamel product. Prior to tooth eruption, enamel proteins are digested by proteases and reabsorbed by ameloblasts. Two extracellular matrix proteases are involved in the cleavage of enamel proteins: matrix metalloproteinase 20 (Mmp-20)² (33) and kallikrein 4 (Klk4) (34).Mmp-20 is secreted along with amelogenin, ameloblastin, and enamelin by secretory stage ameloblasts (35–37). Mmp-20 activity can account for the range of cleavages observed in secretory stage enamel proteins (38) and appears to be the only protease secreted by ameloblasts during the secretory stage. Mmp20-null mice have enamel that is thinner and softer than normal, lacks enamel rod organization, and tends to chip off the crown surface (39, 40). Like the other secretory stage enamel proteins, Mmp20 expression appears to be restricted to developing teeth (41), as is the diseased phenotype when the human gene is defective (42–44).Klk4 is a serine protease that is secreted by transition and maturation stage ameloblasts but is not expressed by secretory stage ameloblasts (45, 46). Klk4 might also be expressed by odontoblasts, the cells that form dentin (47). Klk4 has broad substrate specificity (48, 49) and is capable of activating other proteases (50–52) and protease activated receptors (53, 54). Unlike most proteins secreted by ameloblasts, Klk4 is expressed in other tissues, most notably the prostate (55) and endometrium (56). Much attention has been focused on the potential role of Klk4 in cancers. Klk4 is increased in breast cancer stromal cells (57), in prostate cancer cells (58–61), and ovarian cancer cells (62–65). Despite this focus on the potential role of Klk4 in tumors, very little is known about the normal expression and function of Klk4 in nondental tissues. A loss of function mutation in both human KLK4 alleles caused a hypomaturation enamel phenotype in the absence of any observable defects elsewhere in the body (66).To gain insights into the role of Klk4 in normal dental enamel formation, and to better characterize the normal temporal and spatial patterns of Klk4 expression, we have used gene targeting to knock out normal Klk4 expression, while replacing the Klk4 code with lacZ, the bacterial gene encoding β-galactosidase reporter in mice. We demonstrate that Klk4 is not expressed by secretory stage ameloblasts, but is specifically expressed by ameloblasts later in enamel formation and is necessary for the proper removal of enamel proteins, the final thickening of enamel crystals, and ultimately, for hardening of the enamel layer.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[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]     

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.Gram-negative bacteria have evolved several specialized protein secretion systems to secrete a wide variety of substrate proteins into the extracellular milieu or to inject them into other, often eukaryotic, cells (1). Secreted proteins and their associated secretion systems are very important in bacterial virulence and interactions with other organisms (2). One of the most recent discoveries in this field is the Type VI secretion system (T6SS),¹ which occurs widely across bacterial species (3, 4) and can target proteins to both bacterial and eukaryotic cells (5). The significance of the T6SS is becoming increasingly apparent. It has been implicated in virulence, commensalism, and symbiosis with eukaryotes (5, 6). Additionally, in many bacteria, the T6SS is now implicated in antibacterial activity. T6SS-mediated antibacterial killing appears to be important for competition between bacterial species, for example within the resident microflora of a eukaryotic host (5, 7).Secretion by the T6SS relies on 13 conserved core components which are predicted to form a large machinery associated with the cell envelope, including membrane-bound and bacteriophage tail-like subassemblies (8, 9). The membrane bound subassembly consists of inner membrane proteins (TssLM) and an outer membrane lipoprotein (TssJ) and is anchored to the cell wall. The phage tail-like assembly consists of several proteins that show structural homology with T4 phage tail proteins or are organized in similar structures (10). Hcp (TssD) proteins form hexameric rings and are thought to stack into tube-like structures (11, 12). This Hcp tube is believed to be capped by a trimer of VgrG (TssI) proteins, which share structural homology with the needle of the T4 phage tail (10, 13). In addition, VipA (TssB) and VipB (TssC) form a large tubular structure highly reminiscent of the T4 phage tail sheath (14, 15). Such similarities have led to the idea that the T6SS resembles an inverted contractile bacteriophage infection machinery and injects substrates via an Hcp/VgrG needle into other cells. Recent models propose that the VipA/B sheath surrounds the Hcp/VgrG needle and contraction of the VipA/B tube pushes the Hcp/VgrG needle out of the cell (16–18). It has been postulated that this mechanism can be triggered by close contact with other neighboring cells (19–21).Assembly, localization, and remodelling of VipA/B tubules in vivo depend on the AAA+ ATPase ClpV (TssH), another essential core component of the T6SS (14, 16, 17). ClpV also interacts with the accessory component Fha (TagH) (22, 23), which is found in a subset of T6SSs (4). The Fha protein has an N-terminal domain with a forkhead associated motif, which is predicted to bind phospho-threonine peptides (24). In Pseudomonas aeruginosa, Fha1 is phosphorylated by the Thr/Ser kinase PpkA (TagE) and dephosphorylated by the phosphatase PppA (TagG), and the phosphorylation state of Fha1 regulates the activity of the T6SS (22, 23). Phosphorylation of Fha in P. aeruginosa is also controlled by additional components, which act upstream of PpkA and form a regulatory cascade for T6SS activation (22, 25). Although homologs of PpkA and PppA have been identified in the T6SS gene clusters of certain other bacteria (3), the regulation of the T6SS by post-translational protein phosphorylation has not yet been experimentally investigated outside of Pseudomonas.To understand how the T6SS affects eukaryotic and bacterial cells, it is critical to identify substrate proteins secreted by the T6SS. The VgrG and Hcp proteins were the first identified T6SS substrates and appear to be generally secreted to the external milieu by all T6SSs (26). However, as mentioned above, Hcp and VgrG are core components of the T6SS machinery and therefore represent extracellular components of the secretion apparatus rather than genuine secreted effector proteins. Nonetheless, a limited number of VgrG homologs with extra functional effector domains at the C terminus have been identified or predicted, which account for some of the T6SS dependent effects seen against bacteria and eukaryotes. For example, the C-terminal domain of VgrG-1 from Vibrio cholerae shows actin crosslinking activity in eukaryotic cells (13, 27) and the C-terminal domain of V. cholerae VgrG-3 has bacterial cell wall hydrolase activity (28, 29).Recently, following much effort in the field, a small number of proteins secreted by the T6SS, but not structural components, have been experimentally identified. These proteins are regarded as true secreted substrates of the T6SS, with effector functions in target cells (29–35). For example, antibacterial T6SS-secreted effector proteins with peptidoglycan amidase (cell wall hydrolysis) function, the Type VI amidase effector (Tae) proteins, have been identified in Burkholderia thailandensis (32), P. aeruginosa (31), and Serratia marcescens (30). These Tae proteins play a role in T6SS-mediated antibacterial killing activity and genes encoding four families of Tae protein have been widely identified in other bacteria with T6SSs (32). T6SS-secreted effector proteins which are not peptidoglycan hydrolases have also been reported, including Tse2 secreted by P. aeruginosa, which acts in the bacterial cytoplasm (31), and the VasX and TseL proteins secreted by the V. cholerae T6SS, which are suggested to target membrane lipids (29, 34, 35). In the case of antibacterial T6SSs, the secreting bacterial cells are protected from their own T6SS effector proteins by specific immunity proteins (29–32, 35). However, given the large number of T6SSs in different bacterial species and their apparent ability to secrete multiple substrates, experimentally identified T6-secreted effector proteins still remain surprisingly scarce.Here we report the identification of multiple T6SS-secreted effector proteins in S. marcescens. S. marcescens is an opportunistic pathogen, for example causing ocular infections, nosocomial septicemia and pneumonia (36). Previously, we have identified a T6SS in S. marcescens Db10, which targets and efficiently kills other bacterial cells and plays a role in antibacterial competition (37). We have recently demonstrated that this T6SS secretes two antibacterial effectors, the Tae4 homologs Ssp1 and Ssp2, with cognate immunity proteins Rap1a and Rap2a (30).In this work, we report the analysis of the T6SS-dependent secretome of S. marcescens by label-free quantitation (LFQ) mass spectrometry and describe the identification and characterization of four novel T6SS-secreted effector proteins. These were confirmed as antibacterial toxins and specific immunity proteins were identified. Additionally, this global secretomic analysis, in combination with genetic and phosphoproteomic analyses, demonstrated that a post-translational phosphorylation system influences the ability of the S. marcescens T6SS to secrete effector proteins. Although this system uses homologs of the P. aeruginosa PpkA, PppA and Fha components, the circumstances and impact of Fha phosphorylation were shown to vary between organisms.