Identification of Regions Responsible for the Open Conformation of S100A10 Using Chimaeric S100A11/S100A10 Proteins

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by ANS fluorescence and phenyl Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target-binding site. In contrast, S100A11 chimeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and consequently, unobservable annexin A2 binding. This work represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10

DNA damage-induced translocation of S100A11 into the nucleus regulates cell proliferation

<p>Abstract</p> <p>Background</p> <p>Proteins are able to react in response to distinct stress stimuli by alteration of their subcellular distribution. The stress-responsive protein S100A11 belongs to the family of multifunctional S100 proteins which have been implicated in several key biological processes. Previously, we have shown that S100A11 is directly involved in DNA repair processes at damaged chromatin in the nucleus. To gain further insight into the underlying mechanism subcellular trafficking of S100A11 in response to DNA damage was analyzed.</p> <p>Results</p> <p>We show that DNA damage induces a nucleolin-mediated translocation of S100A11 from the cytoplasm into the nucleus. This translocation is impeded by inhibition of the phosphorylation activity of PKCα. Translocation of S100A11 into the nucleus correlates with an increased cellular p21 protein level. Depletion of nucleolin by siRNA severely impairs translocation of S100A11 into the nucleus resulting in a decreased p21 protein level. Additionally, cells lacking nucleolin showed a reduced colony forming capacity.</p> <p>Conclusions</p> <p>These observations suggest that regulation of the subcellular distribution of S100A11 plays an important role in the DNA damage response and p21-mediated cell cycle control.</p

Unmasking the Annexin I Interaction from the Structure of Apo-S100A11

AbstractS100A11 is a homodimeric EF-hand calcium binding protein that undergoes a calcium-induced conformational change and interacts with the phospholipid binding protein annexin I to coordinate membrane association. In this work, the solution structure of apo-S100A11 has been determined by NMR spectroscopy to uncover the details of its calcium-induced structural change. Apo-S100A11 forms a tight globular structure having a near antiparallel orientation of helices III and IV in calcium binding site II. Further, helices I and IV, and I and I′, form a more closed arrangement than observed in other apo-S100 proteins. This helix arrangement in apo-S100A11 partially buries residues in helices I (P3, E11, A15), III (V55, R58, M59), and IV (A86, C87, S90) and the linker (A45, F46), which are required for interaction with annexin I in the calcium-bound state. In apo-S100A11, this results in a “masked” binding surface that prevents annexin I binding but is uncovered upon calcium binding

Evolutionary origin and diversification of epidermal barrier proteins in amniotes.

The evolution of amniotes has involved major molecular innovations in the epidermis. In particular, distinct structural proteins that undergo covalent cross-linking during cornification of keratinocytes facilitate the formation of mechanically resilient superficial cell layers and help to limit water loss to the environment. Special modes of cornification generate amniote-specific skin appendages such as claws, feathers, and hair. In mammals, many protein substrates of cornification are encoded by a cluster of genes, termed the epidermal differentiation complex (EDC). To provide a basis for hypotheses about the evolution of cornification proteins, we screened for homologs of the EDC in non-mammalian vertebrates. By comparative genomics, de novo gene prediction and gene expression analyses, we show that, in contrast to fish and amphibians, the chicken and the green anole lizard have EDC homologs comprising genes that are specifically expressed in the epidermis and in skin appendages. Our data suggest that an important component of the cornified protein envelope of mammalian keratinocytes, that is, loricrin, has originated in a common ancestor of modern amniotes, perhaps during the acquisition of a fully terrestrial lifestyle. Moreover, we provide evidence that the sauropsid-specific beta-keratins have evolved as a subclass of EDC genes. Based on the comprehensive characterization of the arrangement, exon-intron structures and conserved sequence elements of EDC genes, we propose new scenarios for the evolutionary origin of epidermal barrier proteins via fusion of neighboring S100A and peptidoglycan recognition protein genes, subsequent loss of exons and highly divergent sequence evolution

Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing.

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy

Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen.

The inducible innate immune response to infection requires a concerted process of gene expression that is regulated at multiple levels. Most global analyses of the innate immune response have focused on transcription induced by defined immunostimulatory ligands, such as lipopolysaccharide. However, the response to pathogens involves additional complexity, as pathogens interfere with virtually every step of gene expression. How cells respond to pathogen-mediated disruption of gene expression to nevertheless initiate protective responses remains unclear. We previously discovered that a pathogen-mediated blockade of host protein synthesis provokes the production of specific pro-inflammatory cytokines. It remains unclear how these cytokines are produced despite the global pathogen-induced block of translation. We addressed this question by using parallel RNAseq and ribosome profiling to characterize the response of macrophages to infection with the intracellular bacterial pathogen Legionella pneumophila. Our results reveal that mRNA superinduction is required for the inducible immune response to a bacterial pathogen

Role of genes differentially expressed in thyroid carcinogenesis

Thyroid cancer represents the most common endocrine malignancy, and its incidence has increased significantly over the last few decades. Papillary thyroid carcinoma (PTC), the most frequent neoplasia originating from the thyroid epithelium, accounts for about 80% of all thyroid cancers. PTC is characterized by rearrangements of RET and NTRK1 receptor tyrosine kinases, or by activating point mutations in the BRAF serine/threonine kinase or in the RAS genes. Even though the identification of PTC-associated oncogenes has provided a great contribution to the understanding of PTC pathogenesis, the molecular mechanisms underlying the development of this neoplasia, including the role of tumour suppressor genes, are still far from being completely elucidated. Recently, global gene expression analyses have provided new findings contributing to the dissection of thyroid tumour pathogenesis, through the identification of genes discriminating among different histotypes, candidates as new therapeutic targets and possible tumour suppressor genes. Despite the numerous gene expression studies, there are few data addressing the role of differentially expressed genes in the pathogenesis of thyroid tumours. A microarray gene expression profile previously determined in our laboratory identified a list of genes differentially expressed in PTC versus normal thyroid; among those, we selected TIMP3, S100A11 and CITED1 genes for which a role in the pathogenesis of PTC was also suggested by recently published gene and protein expression data. In this PhD project, we performed functional studies in order to assess the role of TIMP3, S100A11 and CITED1 genes in thyroid carcinogenesis, with the aim of unveiling novel mechanisms and identifying novel therapeutic targets in thyroid tumours. TIMP3 (Tissue Inhibitor of Metalloproteinases-3) is a secreted protein able to inhibit extracellular matrix metalloproteinases. TIMP3 gene promoter has been found hypermethylated in thyroid cancer and its downregulation was associated with several aggressive tumour features. To investigate the role of TIMP3 in the pathogenesis of PTC we used an integrated approach including analysis of several gene expression data sets and functional studies. TIMP3 was found to be downregulated in a consistent fraction in PTCs, with respect to normal thyroid. Restoration of TIMP3 in the PTC-derived NIM1 cell line had no effect on growth rate; however, it reduced migration, invasion and anchorage independent growth. The striking effect was observed in vivo, as TIMP3 reduced the tumourigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. All these observations suggest a tumour suppressor role in thyroid carcinogenesis. S100A11 (calgizzarin) is a member of the S100 Ca2+-binding protein family, which includes at least 20 proteins. Its role in tumour is not well known, and it appears to have distinct functions in different tumour types. Our microarray analysis showed that S100A11 is overexpressed in PTC compared to normal thyroid. In order to study the role of S100A11 in thyroid carcinogenesis, we performed functional in vitro and in vivo analysis. Analysis of cellular localization in PTC-derived K 1 cell line, revealed that S100A11 was mainly cytoplasmic and was able to trans locate into the nucleus after Ca2+ and TGF-β stimulation. We also found that this translocation did not increase p21 level, a negative regulator of cell growth. Moreover, we found that S100A11 did not alter the activation level of the EGF/EGFR pathway. We then investigated the effect of S100A11 silencing on tumourigenic properties of K 1 cells. We found that S100A11 silencing did not affect cell proliferation, but it exerted a role on the anchorage-independent growth. Analysis of mouse tumour xenografts showed that S100A11 was not involved in the in vivo tumourigenicity of K1 cells. Concomitantly, we assessed the effect of S100A11 gene modulation on the transforming potential of PTC-associated oncogenes. Cotransfection experiments in NIH3T3 cells, which represent a useful model for studying in vitro oncogene activity, showed that S100A11 gene was able to enhance the transforming capability of the PTC-associated TRK-T3 oncogene. Stable NIH3T3 foci expressing TRK-T3 and S100A11 concomitantly to TRK -T3 were produced. Functional studies showed that S I OOA 11 was not involved in the regulation of cell proliferation, whereas it was able to promote invasion, anchorage independent growth and in vivo tumourigenicity of these transformed foci. CITED1 (CBP/p300-Interacting Transactivators with glutamic acid (E) and aspartic acid (D)-rich C-terminal domain 1) is part of a family of transcriptional cofactors that regulates diverse CBP/p300 transcriptional responses. In different microarray studies· CITED 1 was found overexpressed in PTC. It was also proposed as a highly sensitive and specific diagnostic marker, useful in differentiating PTC from benign and malignant thyroid tumours. For this reason, we are interested in the functional role of CITED1 in this histotype. Preliminary experiments involved the analysis of CITED 1 expression in a collection of PTC-derived cell lines and the construction of CITED1 cDNA expression vectors. Further analysis will involve the investigation of its effect on transforming activity of PTC-associated oncogenes and the effect of its gene silencing on the phenotype of a PTC-derived cellular model

The Gene Expression Analysis of Blood Reveals S100A11 and AQP9 as Potential Biomarkers of Infective Endocarditis

BACKGROUND: The diagnostic and prognostic assessments of infective endocarditis (IE) are challenging. To investigate the host response during IE and to identify potential biomarkers, we determined the circulating gene expression profile using whole genome microarray analysis. METHODS AND RESULTS: A transcriptomic case-control study was performed on blood samples from patients with native valve IE (n = 39), excluded IE after an initial suspicion (n = 10) at patient's admission, and age-matched healthy controls (n = 10). Whole genome microarray analysis showed that patients with IE exhibited a specific transcriptional program with a predominance of gene categories associated with cell activation as well as innate immune and inflammatory responses. Quantitative real-time RT-PCR performed on a selection of highly modulated genes showed that the expression of the gene encoding S100 calcium binding protein A11 (S100A11) was significantly increased in patients with IE in comparison with controls (P<0.001) and patients with excluded IE (P<0.05). Interestingly, the upregulated expression of the S100A11 gene was more pronounced in staphylococcal IE than in streptococcal IE (P<0.01). These results were confirmed by serum concentrations of the S100A11 protein. Finally, we showed that in patients with IE, the upregulation of the aquaporin-9 gene (AQP9) was significantly associated with the occurrence of acute heart failure (P = 0.02). CONCLUSIONS: Using transcriptional signatures of blood samples, we identified S100A11 as a potential diagnostic marker of IE, and AQP9 as a potential prognostic factor