Molecular evolution of the Thrombospondin superfamily.

Thrombospondins (TSPs) are multidomain, calcium-binding glycoproteins that have wide-ranging roles in vertebrates in cell interactions, extracellular matrix (ECM) organisation, angiogenesis, tissue remodelling, synaptogenesis, and also in musculoskeletal and cardiovascular functions. Land animals encode five TSPs, which assembly co-translationally either as trimers (subgroup A) or pentamers (subgroup B). The vast majority of research has focused on this canonical TSP family, which evolved through the whole-genome duplications that took place early in the vertebrate lineage. With benefit of the growth in genome- and transcriptome-predicted proteomes of a much wider range of animal species, examination of TSPs throughout metazoan phyla has revealed extensive conservation of subgroup B-type TSPs in invertebrates. In addition, these searches established that canonical TSPs are, in fact, one branch within a TSP superfamily that includes other clades designated mega-TSPs, sushi-TSPs and poriferan-TSPs. Despite the apparent simplicity of poriferans and cnidarians as organisms, these phyla encode a greater diversity of TSP superfamily members than vertebrates. We discuss here the molecular characteristics of the TSP superfamily members, current knowledge of their expression profiles and functions in invertebrates, and models for the evolution of this complex ECM superfamily.

Passing the post: roles of posttranslational modifications in the form and function of extracellular matrix.

The extracellular matrix (ECM) is central to the physiology of animal tissues, through its multifaceted roles in tissue structure, mechanical properties, and cell interactions, and by its cell-signaling activities that regulate cell phenotype and behavior. The secretion of ECM proteins typically involves multiple transport and processing steps within the endoplasmic reticulum and the subsequent compartments of the secretory pathway. Many ECM proteins are substituted with various posttranslational modifications (PTMs) and there is increasing evidence of how PTM additions are required for ECM protein secretion or functionality within the extracellular milieu. The targeting of PTM-addition steps may thus offer opportunities to manipulate ECM quality or quantity, in vitro or in vivo. This review discusses selected examples of PTMs of ECM proteins for which the PTM has known importance for anterograde trafficking and secretion of the core protein, and/or loss-of-function of the respectively modifying enzyme leads to alterations of ECM structure or function with pathophysiological consequences in humans. Members of the protein disulfide isomerase (PDI) family have central roles in disulfide bond formation and isomerization within the endoplasmic reticulum, and are discussed in relation to emerging knowledge of the roles of certain PDIs in ECM production in the pathophysiological context of breast cancer. Cumulative data suggest the possible applicability of inhibition of PDIA3 activity to modulate ECM composition and functionality within the tumor microenvironment.

Protein disulfide isomerase A3 activity promotes extracellular accumulation of proteins relevant to basal breast cancer outcomes in human MDA-MB-A231 breast cancer cells.

Metastasis and recurrence of breast cancer remain major causes of patient mortality, and there is an ongoing need to identify new therapeutic targets relevant to tumor invasion. Protein disulfide isomerase A3 (PDIA3) is a disulfide oxidoreductase and isomerase of the endoplasmic reticulum that has known extracellular substrates and has been correlated with aggressive breast cancers. We show that either prior PDIA3 inhibition by the disulfide isomerase inhibitor 16F16 or depletion of heparin-binding proteins strongly reduces the activity of conditioned medium (CM) of MDA-MB-231 human breast cancer cells to support promigratory cell spreading and F-actin organization by newly adherent MDA-MB-231 cells. Quantitative proteomics to investigate effects of 16F16 inhibition on heparin-binding proteins in the CM of MDA-MB-231 cells identified 80 proteins reproducibly decreased at least twofold (at q ≤ 0.05) after 16F16 treatment. By Gene Ontology analysis, many of these have roles in extracellular matrix (ECM) structure and function and cell adhesion; ribosomal proteins that also correlate with extracellular vesicles were also identified. Protein-protein interaction analysis showed that many of the extracellular proteins have known network interactions with each other. The predominant types of disulfide-bonded domains in the extracellular proteins contained ß-hairpin folds, with the knottin fold the most common. From human breast cancer data sets, the extracellular proteins were found to correlate specifically with the basal subtype of breast cancer and their high expression in tumors correlated with reduced distant metastasis-free survival. These data provide new evidence that PDIA3 may be a relevant therapeutic target to alter properties of the ECM-associated microenvironment in basal breast cancer.

PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2.

The matricellular glycoprotein thrombospondin-1 (TSP1) has complex roles in the extracellular matrix (ECM) and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulfide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalize in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3-/- mouse embryo fibroblasts (Pdia3-/- MEFs), led to alterations in the composition of cell-derived extracellular matrix, involving changed abundance of fibronectin and TSP1, was correlated with reduced cell spreading, altered organization of F-actin, and reduced focal adhesions. These cellular phenotypes of Pdia3-/- MEFs were normalized by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEFs. Rescue depended on PDIA3 activity in WT-MEFs and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEFs and Pdia3-/- MEFs identified multiple ECM and growth factor proteins to be downregulated in the CM of Pdia3-/- MEFs. Of these, cell communication network 2 (CCN2) was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3-/- MEFs and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, proadhesive microenvironment, with implications for PDIA3 as a translational target.

In the balance: how do thrombospondins contribute to the cellular pathophysiology of cardiovascular disease?

Thrombospondins (TSPs) are multidomain, secreted proteins that associate with cell surfaces and extracellular matrix. In mammals, there is a large body of data on functional roles of various TSP family members in cardiovascular disease (CVD), including stroke, cardiac remodeling and fibrosis, atherosclerosis, and aortic aneurysms. Coding single nucleotide polymorphisms (SNPs) of TSP1 or TSP4 are also associated with increased risk of several forms of CVD. Whereas interactions and functional effects of TSPs on a variety of cell types have been studied extensively, the molecular and cellular basis for the differential effects of the SNPs remains under investigation. Here, we provide an integrative review on TSPs, their roles in CVD and cardiovascular cell physiology, and known properties and mechanisms of TSP SNPs relevant to CVD. In considering recent expansions to knowledge of the fundamental cellular roles and mechanisms of TSPs, as well as the effects of wild-type and variant TSPs on cells of the cardiovascular system, we aim to highlight knowledge gaps and areas for future research or of translational potential.

Impairment of cell adhesion and migration by inhibition of protein disulphide isomerases in three breast cancer cell lines.

Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified 'scratch wound' assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3-/- mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3-/- MEFs was less effective in promoting cell spreading and F-actin organisation or supporting 'scratch wound' closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

Modelling the early evolution of extracellular matrix from modern Ctenophores and Sponges.

Animals (metazoans) include some of the most complex living organisms on Earth, with regard to their multicellularity, numbers of differentiated cell types, and lifecycles. The metazoan extracellular matrix (ECM) is well-known to have major roles in the development of tissues during embryogenesis and in maintaining homoeostasis throughout life, yet insight into the ECM proteins which may have contributed to the transition from unicellular eukaryotes to multicellular animals remains sparse. Recent phylogenetic studies place either ctenophores or poriferans as the closest modern relatives of the earliest emerging metazoans. Here, we review the literature and representative genomic and transcriptomic databases for evidence of ECM and ECM-affiliated components known to be conserved in bilaterians, that are also present in ctenophores and/or poriferans. Whereas an extensive set of related proteins are identifiable in poriferans, there is a strikingly lack of conservation in ctenophores. From this perspective, much remains to be learnt about the composition of ctenophore mesoglea. The principal ECM-related proteins conserved between ctenophores, poriferans, and bilaterians include collagen IV, laminin-like proteins, thrombospondin superfamily members, integrins, membrane-associated proteoglycans, and tissue transglutaminase. These are candidates for a putative ancestral ECM that may have contributed to the emergence of the metazoans.

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular α-DG associated noncovalently with a transmembrane ß-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (α-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the α-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind α-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for α-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic α-DG non-binders such as laminin-α3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of α-DG, although it appears that this alone is insufficient to mediate significant α-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with α-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

Emergence of a Thrombospondin Superfamily at the Origin of Metazoans.

Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity; however, the repertoire of ECM proteins in nonbilaterians remains unclear. Thrombospondins (TSPs) are known to be well conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highly conserved C-terminal region of TSPs, we identify undisclosed new families of TSP-related proteins in metazoans, designated mega-TSP, sushi-TSP, and poriferan-TSP, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-TSPs, the only form identified in ctenophores, are typically >2,700 aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM, Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis, with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult Nematostella vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly defined TSP superfamily by gene duplications, radiation, and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.

Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction.

Thrombospondins (TSPs) are multidomain glycoproteins with complex matricellular functions in tissue homeostasis and remodeling. We describe a novel role of TSP as a Wnt signaling target in the basal eumetazoan Hydra. Proteome analysis identified Hydra magnipapillata TSP (HmTSP) as a major component of the cnidarian mesoglea. In general, the domain organization of cnidarian TSPs is related to the pentameric TSPs of bilaterians, and in phylogenetic analyses cnidarian TSPs formed a separate clade of high sequence diversity. HmTSP expression in polyps was restricted to the hypostomal tip and tentacle bases that harbor Wnt-regulated organizer tissues. In the hypostome, HmTSP- and Wnt3-expressing cells were identical or in close vicinity to each other, and regions of ectopic tentacle formation induced by pharmacological ß-Catenin activation (Alsterpaullone) corresponded to foci of HmTSP expression. Chromatin immunoprecipitation (ChIP) confirmed binding of Hydra TCF to conserved elements in the HmTSP promotor region. Accordingly, ß-Catenin knockdown by siRNAs reduced normal HmTSP expression at the head organizer. In contrast, knockdown of HmTSP expression led to increased numbers of ectopic organizers in Alsterpaullone-treated animals, indicating a negative regulatory function. Our data suggest an unexpected role for HmTSP as a feedback inhibitor of Wnt signaling during Hydra body axis patterning and maintenance.