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Summary: Embigin (Gp70), a receptor for fibronectin and an ancillary protein for monocarboxylate transporters, is known to regulate stem cell niches in sebaceous gland and bone marrow. Here, we show that embigin expression is at high level during early mouse embryogenesis and that embigin is essential for lung development. Markedly increased neonatal mortality of Emb−/− mice can be explained by the compromised lung maturation: in Emb−/− mice (E17.5) the number and the size of the small airways and distal airspace are significantly smaller, there are fewer ATI and ATII cells, and the alkaline phosphatase activity in amniotic fluid is lower. Emb−/− lungs show less peripheral branching already at E12.5, and embigin is highly expressed in lung primordium. Thus, embigin function is essential at early pseudoglandular stage or even earlier. Furthermore, our RNA-seq analysis and Ki67 staining results support the idea that the development of Emb−/− lungs is rather delayed than defected.

IntroductionProtein arginine deiminases (PADs) are intracellular enzymes that may, especially in pathological conditions, also citrullinate extracellular substrates, including matrisome proteins such as structural proteins in extracellular matrix (ECM). PADs are abundantly expressed in human cancer cells. Citrullination of matrisome proteins has been reported in colon cancer but the phenomenon has never been systematically studied.MethodsTo gain a broader view of citrullination of matrisome proteins in cancer, we analyzed cancer proteomics data sets in 3 public databases for citrullinated matrisome proteins. In addition, we used three-dimensional cell cocultures of fibroblasts and cancer cells and analyzed citrullination of ECM.Results and discussionOur new analysis indicate that citrullination of ECM occurs in human cancer, and there is a significant variation between tumors. Most frequently citrullinated proteins included fibrinogen and fibronectin, which are typically citrullinated in rheumatoid inflammation. We also detected correlation between immune cell marker proteins, matrix metalloproteinases and ECM citrullination, which suggests that in cancer, citrullination of matrisome proteins is predominantly an inflammation-related phenomenon. This was further supported by our analysis of three-dimensional spheroid co-cultures of nine human cancer cell lines and fibroblasts by mass spectrometry, which gave no evidence that cancer cells or fibroblasts could citrullinate matrisome proteins in tumor stroma. It also appears that in the spheroid cultures, matrisome proteins are protected from citrullination.

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

Abstract We report the extent, specific sites and structural requirements of joint inflammation related citrullination in extracellular proteins. A total of 40 synovial fluid samples derived from chronically inflamed human joints were analysed by heparin-agarose fractionation and LC-MS/MS. Citrullination of 55 arginines in extracellular proteins was detected. Importantly, 20% of the sites have a characterized function related to the hallmarks of destructive joint inflammation. E.g. four arginine residues, shown here to be citrullinated, are also affected by mutations in inherited diseases causing haemolysis or blood clotting dysfunction. Citrullination of integrin ligands was selected for further studies since fibronectin R234 in isoDGR was among the most frequently citrullinated arginines in synovial fluid. Assays with synovial fibroblasts and integrin αVβ3 indicated decreased affinity to the enzymatically citrullinated integrin binding sites. To conclude, our data indicate that in inflamed joints extensive citrullination affects the functional arginine residues in extracellular proteins.

Abstract Conformational activation of integrins is generally required for ligand binding and cellular signalling. However, we have previously reported that the nonactivated conformation of α2β1 integrin can also bind to large ligands, such as human echovirus 1. In this study, we show that the interaction between the nonactivated integrin and a ligand resulted in the activation of focal adhesion kinase (FAK) in a protein kinase C dependent manner. A loss-of-function mutation, α2E336A, in the α2-integrin did not prevent the activation of FAK, nor did EDTA-mediated inactivation of the integrin. Full FAK activation was observed, since phosphorylation was not only confirmed in residue Y397, but also in residues Y576/7. Furthermore, initiation of downstream signaling by paxillin phosphorylation in residue Y118 was evident, even though this activation was transient by nature, probably due to the lack of talin involvement in FAK activation and the absence of vinculin in the adhesion complexes formed by the nonactivated integrins. Altogether these results indicate that the nonactivated integrins can induce cellular signaling, but the outcome of the signaling differs from conventional integrin signaling.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.

Long non-coding RNAs (lncRNAs) regulate various cellular processes, and they have emerged as potential biomarkers and therapeutic targets in cancer. We have previously characterized the oncogenic role of lncRNA PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA) in cutaneous squamous cell carcinoma (cSCC), the most common metastatic skin cancer. In this study, we show that knockdown of PICSAR in cSCC cells upregulates expression of α2, α5 and β1 integrins, resulting in increased cell adhesion and decreased cell migration on collagen I and fibronectin. In contrast, overexpression of PICSAR in cSCC cells downregulates expression of α2, α5 and β1 integrins on cell surface, resulting in decreased cell adhesion on collagen I and fibronectin and increased cell migration. These results demonstrate a novel mechanism for regulation of the expression of collagen and fibronectin binding integrins by lncRNA PICSAR, leading to altered adhesion and migration of cSCC cells. This article has an associated First Person interview with the first author of the paper.

Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future.

Half of the 18 human integrins α subunits have an inserted αI domain yet none have been observed in species that have diverged prior to the appearance of the urochordates (ascidians). The urochordate integrin αI domains are not human orthologues but paralogues, but orthologues of human αI domains extend throughout later-diverging vertebrates and are observed in the bony fish with duplicate isoforms. Here, we report evidence for orthologues of human integrins with αI domains in the agnathostomes (jawless vertebrates) and later diverging species. Sequence comparisons, phylogenetic analyses and molecular modeling show that one nearly full-length sequence from lamprey and two additional fragments include the entire integrin αI domain region, have the hallmarks of collagen-binding integrin αI domains, and we show that the corresponding recombinant proteins recognize the collagen GFOGER motifs in a metal dependent manner, unlike the α1I domain of the ascidian C. intestinalis. The presence of a functional collagen receptor integrin αI domain supports the origin of orthologues of the human integrins with αI domains prior to the earliest diverging extant vertebrates, a domain that has been conserved and diversified throughout the vertebrate lineage.

Cancer-testis (CT) antigens are predominantly expressed in testis or placenta, but absent in most adult tissues. During malignant transformation CT genes are often activated. CT antigen 16 (CT16, PAGE5) is frequently expressed in advanced melanoma but its biological function has been unknown. To examine the role of CT16 in cell survival we knocked it down in A2058 melanoma cells using specific siRNAs and exposed the cells to cancer drug cisplatin known to induce apoptosis. As a result, cell survival was markedly decreased. To study the effects of CT16 on cell survival in more detail, the cellular gene expression profiles were investigated after CT16 silencing in CT16 positive A2058 melanoma cells, as well as after CT16 overexpression in CT16 negative WM-266-4 melanoma cells. Among the 11 genes both upregulated by CT16 silencing and downregulated by CT16 overexpression or vice versa, 4 genes were potentially apoptotic or antiapoptotic genes. CT16 was recognized as a positive regulator of antiapoptotic metallothionein 2A and interleukin 8 genes, whereas it inhibited the expression of apoptosis inducing dickkopf 1 (DKK1) gene. In addition CT16 enhanced the expression of fatty acid binding protein 7, a known promoter of melanoma progression. The effect of CT16 on DKK1 expression was p53 independent. Furthermore, CT16 did not regulate apoptotic genes via DNA methylation. In twenty melanoma metastasis tissue samples average DKK1 mRNA level was shown to be significantly (p

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca(2+)-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca(2+)-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found.

BACKGROUND: The Cancer-Testis antigens (CTA) are proteins expressed in human germ line and certain cancer cells. CTAs form a large gene family, representing 10% of X-chromosomal genes. They have high potential for cancer-specific immunotherapy. However, their biological functions are currently unknown. Prostate associated genes (PAGE) are characterized as CTAs. PAGE5 is one of six proteins belonging to this protein family, also called CT16. METHODOLOGY/PRINCIPAL FINDINGS: In this study we show, using bioinformatics, chromatographic and solution state NMR spectroscopic methods, that PAGE5 is an intrinsically disordered protein (IDP). CONCLUSION/SIGNIFICANCE: The study stands out as the first time structural characterization of the PAGE family protein and introduces how solution state NMR spectroscopy can be effectively utilized for identification of molecular recognition regions (MoRF) in IDPs, known often as transiently populated secondary structures.

Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA. [ABSTRACT FROM AUTHOR]