The biochemical, biological, and pathological kaleidoscope of cell surface substrates processed by matrix metalloproteinases

Matrix metalloproteinases (MMPs) constitute a family of more than 20 endopeptidases. Identification of specific matrix and non-matrix components as MMP substrates showed that, aside from their initial role as extracellular matrix modifiers, MMPs play significant roles in highly complex processes such as the regulation of cell behavior, cell-cell communication, and tumor progression. Thanks to the comprehensive examination of the expanded MMP action radius, the initial view of proteases acting in the soluble phase has evolved into a kaleidoscope of proteolytic reactions connected to the cell surface. Important classes of cell surface molecules include adhesion molecules, mediators of apoptosis, receptors, chemokines, cytokines, growth factors, proteases, intercellular junction proteins, and structural molecules. Proteolysis of cell surface proteins by MMPs may have extremely diverse biological implications, ranging from maturation and activation, to inactivation or degradation of substrates. In this way, modification of membrane-associated proteins by MMPs is crucial for communication between cells and the extracellular milieu, and determines cell fate and the integrity of tissues. Hence, insights into the processing of cell surface proteins by MMPs and the concomitant effects on physiological processes as well as on disease onset and evolution, leads the way to innovative therapeutic approaches for cancer, as well as degenerative and inflammatory diseases.     

The Biochemical,Biological, and Pathological Kaleidoscope of Cell Surface Substrates Processed by Matrix Metalloproteinases

ABSTRACT

Matrix metalloproteinases (MMPs) constitute a family of more than 20 endopeptidases. Identification of specific matrix and non-matrix components as MMP substrates showed that, aside from their initial role as extracellular matrix modifiers, MMPs play significant roles in highly complex processes such as the regulation of cell behavior, cell-cell communication, and tumor progression. Thanks to the comprehensive examination of the expanded MMP action radius, the initial view of proteases acting in the soluble phase has evolved into a kaleidoscope of proteolytic reactions connected to the cell surface. Important classes of cell surface molecules include adhesion molecules, mediators of apoptosis, receptors, chemokines, cytokines, growth factors, proteases, intercellular junction proteins, and structural molecules. Proteolysis of cell surface proteins by MMPs may have extremely diverse biological implications, ranging from maturation and activation, to inactivation or degradation of substrates. In this way, modification of membrane-associated proteins by MMPs is crucial for communication between cells and the extracellular milieu, and determines cell fate and the integrity of tissues. Hence, insights into the processing of cell surface proteins by MMPs and the concomitant effects on physiological processes as well as on disease onset and evolution, leads the way to innovative therapeutic approaches for cancer, as well as degenerative and inflammatory diseases.     

Transferring intercellular signals and traits between cancer cells: extracellular vesicles as “homing pigeons”

Extracellular vesicles are cell-derived vesicles, which can transport various cargos out of cells. From their cell of origin, the content molecules (proteins, non-coding RNAs including miRNAs, DNA and others) can be delivered to neighboring or distant cells and as such extracellular vesicles can be regarded as vehicles of intercellular communication or “homing pigeons”. Extracellular vesicle shuttling is able to actively modulate the tumor microenvironment and can partake in tumor dissemination. In various diseases, including cancer, levels of extracellular vesicle secretion are altered resulting in different amounts and/or profiles of detectable vesicular cargo molecules and these distinct content profiles are currently being evaluated as biomarkers. Apart from their potential as blood-derived containers of specific biomarkers, the transfer of extracellular vesicles to surrounding cells also appears to be involved in the propagation of phenotypic traits. These interesting properties have put extracellular vesicles into the focus of many recent studies.Here we review findings on the involvement of extracellular vesicles in transferring traits of cancer cells to their surroundings and briefly discuss new data on oncosomes, a larger type of vesicle. A pressing issue in cancer treatment is rapidly evolving resistance to many initially efficient drug therapies. Studies investigating the role of extracellular vesicles in this phenomenon together with a summary of the technical challenges that this field is still facing, are also presented. Finally, emerging areas of research such as the analysis of the lipid composition on extracellular vesicles and cutting-edge techniques to visualise the trafficking of extracellular vesicles are discussed.     

“Treasure your exceptions”: recent advances in molecular genetics of glomerular disease

The glomerular filtration barrier consists of endothelial cells, the glomerular basement membrane, and podocytes. The membrane is a highly crosslinked macromolecular meshwork composed of specific extracellular matrix proteins. The adjacent foot processes of podocytes are bridged along their basolateral surfaces by a slit diaphragm (a porous filter structure of nephrin molecules). Recent discoveries of mutations in the range of genes encoding proteins involved in the structure or function of the glomerular filtration barrier have provided new insights into mechanisms of glomerular diseases. In this review, we summarize recent progress in the elucidation of the genetic basis of some glomerulopathies in humans.     

Integrins and other cell adhesion molecules

Cell-cell and cell-substratum interactions are mediated through several different families of receptors. In addition to targeting cell adhesion to specific extracellular matrix proteins and ligands on adjacent cells, these receptors influence many diverse processes including cellular growth, differentiation, junction formation, and polarity. Several families of adhesion receptors have been identified. These include: 1) the integrins, heterodimeric molecules that function both as cell-substratum and cell-cell adhesion receptors; 2) the adhesion molecules of the immunoglobulin superfamily, which are involved in cell-cell adhesion and especially important during embryo-genesis, wound healing, and the inflammatory response; 3) the cadherins, developmentally regulated, calcium-dependent homophilic cell-cell adhesion proteins; 4) the LEC-CAMs, cell adhesion molecules with lectin-like domains that mediate white blood cell/endothelial cell adhesion; and 5) homing receptors that target lymphocytes to specific lymphoid tissue. In this review we summarize recent data describing the structure and function of some of these cell adhesion molecules (with special emphasis on the integrin family) and discuss the possible role of these molecules in development, inflammation, wound healing, coagulation, and tumor metastasis.     

Emerging roles of CCN proteins in vascular development and pathology

The CCN family of proteins consists of 6 members (CCN1-CCN6) that share conserved functional domains. These matricellular proteins interact with growth factors, extracellular matrix (ECM) proteins, cell surface integrins and other receptors to promote ECM-intracellular signaling. This signaling leads to propagation of a variety of cellular actions, including adhesion, invasion, migration and proliferation within several cell types, including epithelial, endothelial and smooth muscle cells. Though CCNs share significant homology, the function of each is unique due to distinct and cell specific expression patterns. Thus, their correct spatial and temporal expressions are critical during embryonic development, wound healing, angiogenesis and fibrosis. Disruption of these patterns leads to severe development disorders and contributes to the pathological progression of cancers, vascular diseases and chronic inflammatory diseases such as colitis, rheumatoid arthritis and atherosclerosis. While the effects of CCNs are diverse, this review will focus on the role of CCNs within the vasculature during development and in vascular diseases.     

Proteomic analysis of mammalian primary cilia

The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium-shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia-specific proteins. These possible primary cilia-specific proteins include EVC2, INPP5E, and inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.     

The echinoderm adhesome

Although the development of sea urchin embryos has been studied extensively and clearly involves both cell adhesion and cell migration, rather little is known about the adhesion receptors and extracellular matrix molecules involved. The completion of the genome of Strongylocentrotus purpuratus allows a comprehensive survey of the complement of cell-cell and cell-matrix adhesion molecules in this organism. Furthermore, the phylogenetic position of echinoderms offers the opportunity to compare the complement of adhesion proteins between protostome and deuterostome invertebrates and between invertebrate and vertebrate deuterostomes. Many aspects of development and cell interactions differ among these different taxa and it is likely that analysis of the spectrum of adhesion receptors and extracellular matrix proteins can open up new insights into which molecules have evolved to suit particular developmental processes. In this paper, we report the results of an initial analysis along these lines. The echinoderm adhesome (complement of adhesion-related genes/proteins) is similar overall to that of other invertebrates although there are significant deuterostome-specific innovations and some interesting features previously thought to be chordate or vertebrate specific.     

Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha

The protocadherin-alpha (CNR/Pcdhalpha) and protocadherin-gamma (Pcdhgamma) proteins, members of the cadherin superfamily, are putative cell recognition/adhesion molecules in the brain. Overexpressed cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines, although hardly any overexpressed CNR/Pcdhalpha proteins are expressed on the cell surface, except on HEK293T cells, which show low expression. We analyzed the expression of CNR/Pcdhalpha and Pcdhgamma in HEK293T cells and found that they formed a protein complex and that Pcdhgamma enhanced the surface expression of CNR/Pcdhalpha. This enhanced surface expression was confirmed by flow cytometry analysis and by marking cell surface proteins with biotin. The enhancement was observed using different combinations of CNR/Pcdhalpha and Pcdhgamma proteins. The surface expression activity was enhanced by the extracellular domains of the proteins, which could bind each other. Their cytoplasmic domains also had binding activity and influenced their localization. Their protein-protein interaction was also detected in extracts of mouse brain and two neuroblastoma cell lines. Thus, interactions between CNR/Pcdhalpha and Pcdhgamma regulate their surface expression and contribute to the combinatorial diversity of cell recognition proteins in the brain.     

Augmentation of RANTES-induced extracellular signal-regulated kinase mediated signaling and T cell adhesion by elastase-treated fibronectin

T cells migrating across extracellular matrix (ECM) barriers toward their target, the inflammatory site, should respond to chemoattractant cytokines and to the degradation of ECM by specific enzymes. In this study, we examined the effects of RANTES and ECM proteins treated with human leukocyte elastase on T cell activation and adhesion to the ECM. We found that human peripheral blood T cells briefly suspended with RANTES (0.1-100 ng/ml) had increased phosphorylation of their intracellular extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase involved in the activation of several intracellular downstream effector molecules implicated in cell adhesion and migration. Consequently, a small portion (12-20%) of the responding cells adhered to fibronectin (FN). However, when the T cells were exposed to RANTES in the presence of native immobilized FN, laminin, or collagen type I, ERK phosphorylation was partially inhibited, suggesting that this form of the ECM proteins can down-regulate RANTES-induced intracellular signaling. In contrast, when the T cells were exposed to RANTES in the presence of elastase-treated immobilized FN, but not to elastase-treated laminin, ERK phosphorylation was markedly increased. Furthermore, a large percentage (30%) of RANTES-activated T cells adhered to the enzymatically treated FN in a beta1 integrin-dependent fashion. Thus, while migrating along chemotactic gradients within the ECM, T cells can adapt their adhesive performance according to the level of cleavage induced by enzymes to the matrix.     

Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells

Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology. From 965 unique peptides carrying biotin labels, we assigned 324 proteins including 235 proteins that have putative signal sequences and/or transmembrane segments. Receptors, transporters, and cell adhesion molecules were the major classes of proteins identified. Besides known cell surface markers of embryonic stem cells, such as alkaline phosphatase, the analysis identified 59 clusters of differentiation-related molecules and more than 80 components of multiple cell signaling pathways that are characteristic of a number of different cell lineages. We identified receptors for leukemia-inhibitory factor, interleukin 6, and bone morphogenetic protein, which play critical roles in the maintenance of undifferentiated mouse embryonic stem cells. We also identified receptors for growth factors/cytokines, such as fibroblast growth factor, platelet-derived growth factor, ephrin, Hedgehog, and Wnt, which transduce signals for cell differentiation and embryonic development. Finally we identified a variety of integrins, cell adhesion molecules, and matrix metalloproteases. These results suggest that D3 cells express diverse cell surface proteins that function to maintain pluripotency, enabling cells to respond to various external signals that initiate differentiation into a variety of cell types.     

Matrix biology meets toxinology

Venoms are cocktails containing pharmacologically active compounds, which drastically affect essential functions of the neuromuscular and cardiovascular system, as well as of blood, kidney and other organs. As the extracellular matrix and its contacts with cells are responsible for maintaining the integrity and functionality of these organs and tissues, it is not surprising that several venom components target matrix molecules and their respective cellular receptors. Many venom components, such as matrix-degrading enzymes, disintegrins, and C-type lectin-like proteins, have been identified and have laid the foundation for the frontier research field of matrix toxinology. Interestingly, many toxins consist of domains which are structurally homologous to modules and domains of matrix proteins, their proteinases and cellular receptors. In addition to finding new agents and tools, which specifically interact with matrix molecules and their receptors, the characterization of known matrix-targeting toxins will provide insights into their molecular modes of action and thus may lead to potential new therapeutic strategies for treating matrix-related diseases, such as blood clotting and thrombocyte-mediated disorders, but also tumor malignancies.     

Dynamic length regulation of sensory stereocilia

Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Studies on actin turnover in stereocilia, as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function, provide new insights into the mechanisms and molecules involved in stereocilia length regulation and long-term maintenance. Comparisons of ongoing investigations on stereocilia with studies on other actin protrusions offer new opportunities to further understand common principles for length regulation, the diversity of its mechanisms, and how the specific needs of each cell are met.     

Galectins as modulators of cell adhesion

The galectins are a family of carbohydrate-binding proteins that are distributed widely in metazoan organisms. Each galectin exhibits a specific pattern of expression in various cells and tissues, and expression is often closely regulated during development. Although these proteins are found mainly in the cell cytoplasm, some are secreted from cells and interact with appropriately glycosylated proteins at the cell surface or within the extracellular matrix. These receptors include cell-adhesion molecules such as integrins, and matrix glycoproteins such as laminin and fibronectin isoforms. Recent studies have increased understanding of the roles of the galectins in regulating cell-cell and cell-matrix adhesion. These interactions are critically involved in modulation of normal cellular motility and polarity and during tissue formation, and loss of adhesive function is implicated in several disease states including tumour progression, inflammation and cystic development in branching epithelia such as kidney tubules. This review discusses recent progress in defining the specificities and mechanisms of action of secreted galectins as multifunctional cell regulators.     

Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice

To address the mechanisms of tolerance to extrathymic proteins, we have generated transgenic mice expressing the lymphocytic choriomeningitis viral (LCMV) glycoprotein (GP) in the beta islet cells of the pancreas. The fate of LCMV GP-specific T cells was followed by breeding the GP transgenic mice with T cell receptor transgenic mice, specific for LCMV and H-2Db. These studies suggest that "peripheral tolerance" of self-reactive T cells does not involve clonal deletion, clonal anergy, or a decrease in the density of T cell receptors or accessory molecules. Instead, this model indicates that self-reactive cytotoxic T cells may remain functionally unresponsive, owing to a lack of appropriate T cell activation. Infection of transgenic mice with LCMV readily abolishes peripheral unresponsiveness to the self LCMV GP antigen, resulting in a CD8+ T cell-mediated diabetes. These data suggest that similar mechanisms may operate in several so-called "T cell-mediated autoimmune diseases."     

Type IV secretion systems and their effectors in bacterial pathogenesis

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.     

A method for production of recombinant mCD1d protein in insect cells

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.     

A non-receptor-mediated mechanism for internalization of molecular chaperones

The evolving realization that stress proteins, which have for many years been considered to be exclusively intracellular molecules under normal conditions, can be released from viable cells via a number of potential routes/pathways has prompted interest into their extracellular biology and intercellular signaling properties. That the stress proteins Hsp60, Hsp70 and gp96 can elicit both pro- and anti-inflammatory effects suggests that these molecules play a key role in the maintenance of immunological homeostasis, and a better understanding of the immunobiology of extracellular stress proteins might reveal new and more effective approaches for controlling and managing infectious disease, inflammatory disease and cancer. A number of cell surface receptors for stress proteins have been identified, and the intracellular consequences of these cell surface receptor-ligand interactions have been characterized. To date, studies into the intercellular signaling properties of stress proteins and their interactions with antigen presenting cells have focused on specific receptor-mediated uptake, and have not considered the fact that such cells can also take up proteins via non-specific endocytosis/pinocytosis. Herein we present a methodological approach for assessing receptor-mediated and non-receptor-mediated uptake of gp96 by rat bone marrow-derived dendritic cells.     

Characterization of proteins secreted during maize microspore culture: arabinogalactan proteins (AGPs) stimulate embryo development

To study molecules secreted from cultured plant cells that promote development, maize microspores were transferred into culture and the conditioned media were collected over time and analysed. Electrophoresis indicated that both non-glycosylated and glycosylated proteins including arabinogalactan proteins (AGPs) appeared in the medium and their concentration increased during the time of culture. The development of embryos was correlated with the presence of specific extracellular proteins, using an experimental system based on a tunicamycin inhibition test. In addition, a precise protein analysis was conducted using MALDI-TOF and ESI-MS-MS techniques. These approaches have allowed the identification of 5 other types of proteins: a cell wall invertase, two thaumatin isoforms, one 1-3 beta-glucanase and two chitinase isoforms. Altogether these experiments and results open ways for research aimed at understanding which molecules stimulate embryo formation. Moreover, AGPs may be used to stimulate the development of microspores (pollen embryogenesis) prepared from non-responsive genotypes.