Analysis on conservation of disulphide bonds and their structural features in homologous protein domain families

International audienceBackground: Disulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue.Results: Using a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues.Conclusion: We observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function

A Computational Approach Identifies Immunogenic Features of Prognosis in Human Cancers

A large number of tumor intrinsic and extrinsic factors determine long-term survival in human cancers. In this study, we stratified 9120 tumors from 33 cancers with respect to their immune cell content and identified immunogenomic features associated with long-term survival. Our analysis demonstrates that tumors infiltrated by CD8+ T cells expressing higher levels of activation marker (PD1hi) along with TCR signaling genes and cytolytic T cell markers (IL2hi/TNF-αhi/IFN-γhi/GZMA-Bhi) extend survival, whereas survival benefit was absent for tumors infiltrated by anergic and hyperexhausted CD8+ T cells characterized by high expression of CTLA-4, TIM3, LAG3, and genes linked to PI3K signaling pathway. The computational approach of using robust and highly specific gene expression signatures to deconvolute the tumor microenvironment has important clinical applications, such as selecting patients who will benefit from checkpoint inhibitor treatment

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 235–241, 2008. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57899/1/20899_ftp.pd

Raman spectroscopy can discriminate between normal, dysplastic and cancerous oral mucosa: a tissue-engineering approach.

Head and neck cancer (HNC) is the sixth most common malignancy worldwide. Squamous cell carcinoma, the primary cause of HNC, evolves from normal epithelium through dysplasia before invading the connective tissue to form a carcinoma. However, less than 18% of suspicious oral lesions progress to cancer, with diagnosis currently relying on histopathological evaluation, which is invasive and time consuming. A non-invasive, real-time, point-of-care method could overcome these problems and facilitate regular screening. Raman spectroscopy is a non-invasive optical technique with the ability to extract molecular level information to help determine the functional groups present in a tissue and the molecular conformations of tissue constituents. In the present study, Raman spectroscopy was assessed for its ability to discriminate between normal, dysplastic and HNC. Tissue engineered models of normal, dysplastic and HNC were constructed using normal oral keratinocytes, dysplastic and HNC cell lines, and their biochemical content predicted by interpretation of spectral characteristics. Spectral differences were evident in both the fingerprint (600/cm to 1800/cm) and high wave-number compartments (2800/cm to 3400/cm). Visible differences were seen in peaks relating to lipid content (2881/cm), protein structure (amide I, amide III), several amino acids and nucleic acids (600/cm to 1003/cm). Multivariate data analysis algorithms successfully identified subtypes of dysplasia and cancer, suggesting that Raman spectroscopy not only has the potential to differentiate between normal, pre-malignant and cancerous tissue models but could also be sensitive enough to detect subtypes of dysplasia or cancer on the basis of their subcellular differences. Copyright © 2016 John Wiley & Sons, Ltd

Micro-Raman Spectroscopy and Univariate Analysis for Monitoring Disease Follow-Up

Micro-Raman spectroscopy is a very promising tool for medical applications, thanks to its sensitivity to subtle changes in the chemical and structural characteristics of biological specimens. To fully exploit these promises, building a method of data analysis properly suited for the case under study is crucial. Here, a linear or univariate approach using a R2 determination coefficient is proposed for discriminating Raman spectra even with small differences. The validity of the proposed approach has been tested using Raman spectra of high purity glucose solutions collected in the 600 to 1,600 cm−1 region and also from solutions with two known solutes at different concentrations. After this validation step, the proposed analysis has been applied to Raman spectra from oral human tissues affected by Pemphigus Vulgaris (PV), a rare life-threatening autoimmune disease, for monitoring disease follow-up. Raman spectra have been obtained in the wavenumber regions from 1,050 to 1,700 cm−1 and 2,700 to 3,200 cm−1 from tissues of patients at different stages of pathology (active PV, under therapy and PV in remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage have been detected at 1,150–1,250 cm−1 (amide III) and 1,420–1,450 cm−1 (CH3 deformation) regions and around 1,650 cm−1 (amide I) and 2,930 cm−1 (CH3 symmetric stretch). The analysis of tissue Raman spectra by the proposed univariate method has allowed us to effectively differentiate tissues at different stages of pathology

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Caractérisation génomique, structurale et fonctionnelle des protéines liant les molécules odorantes dans le système olfactif des moustiques vecteurs de maladies infectieuses

The role of odorant binding proteins in the olfaction of mosquitoes, the primary mechanism of human host recognition, has been an important focus of biological research in the field of infectious disease transmission by these insects. This thesis provides an in depth knowledge of these proteins in three mosquito species Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. A large scale analysis on these genomes has been carried out towards the identification of the odorant binding proteins in the mosquito genomes. Identification of many new OBP members, in particular in the Aedes aegypti and Culex quinquefasciatus species, and an extensive phylogenetic analysis presenting a novel classification of the OBP subfamilies of these mosquito species has been proposed. This results further demonstrates the extraordinary multiplicity and diversity of the OBP gene repertoire in these three mosquito genomes and highlights the striking sequence features that are nevertheless highly conserved across all mosquito OBPs. Owing to the availability of homologous structures from mosquitoes or related species, the 3D structure modelling of all the Classic OBPs from the three genomes (representing in total 137 structures) has been performed. This was completed by large scale docking studies on these structures by screening a large set of compounds that are known to be mosquito attractants or repellents. These provide many exciting new insights into the structural and functional aspects towards understanding the efficacy of some repellents and of some attractants from human emanations. Through molecular dynamics simulation, the structural changes observed in an OBP bounded to an odorant when pH conditions are modified were characterized and the probable mechanism of ligand binding and release is presented. This work provides the first insights to many of the long awaited questions on the genomic, structural and functional characterization of mosquito OBPs and can be viewed as a reliable starting point for further experimental research focussed on these aspects.Dans le système olfactif des moustiques, les protéines liants les molécules odorantes ou odorant binding proteins (OBPs) interviennent dans les toutes premières étapes permettant d'aboutir à la reconnaissance de leurs hôtes et font l'objet d'un intérêt croissant dans les recherches sur la transmission des maladies infectieuses par ces insectes. Le travail présenté a pour objet d'approfondir les connaissances sur ces OBPs dans trois génomes de moustiques, tous vecteurs de maladies infectieuses : Anopheles gambiae, Aedes aegypti et Culex quinquefasciatus. Une analyse à l'échelle de ces génomes a été réalisée et a permis d'identifier un nombre important de nouveaux gènes d'OBPs notamment chez les espèces de moustiques Aedes aegypti et Culex quinquefasciatus. Complétée par une étude phylogénétique du répertoire complet de ces gènes dans les trois génomes étudiés, cette analyse a permis d'établir une nouvelle classification des sous familles des OBPs. Ce résultat démontre l'extraordinaire multiplicité et diversité des gènes impliqués dans l'olfaction chez ces espèces de moustiques tout en mettant en lumière certaines propriétés des séquences des OBPs qui sont hautement conservés chez les moustiques. Grâce à la disponibilité de certaines structures d'OBPs de moustiques ou d'autres insectes apparentées, des modèles structuraux de tous les OBPs de la sous famille dites Classic dans les trois génomes, soit au total 137 structures, ont été construits. Ces structures ont servi de base pour le criblage à grande échelle par docking moléculaire d'une chimiothèque de 126 molécules odorantes connues pour leurs propriétés attractives ou répulsives vis-à-vis des moustiques. Ces résultats fournissent pour la première fois, les bases structurales et fonctionnelles pour la compréhension au niveau moléculaire de l'efficacité de certains agents répulsifs tout comme de l'attractivité de certains agents provenant des émanations humaines. Par simulation de dynamique moléculaire, les changements qui s'opèrent dans une de ces OBPs lorsque celle ci, liée à une molécule odorante, se retrouve dans des conditions de pH modifiée ont été caractérisée et un mécanisme probable par lequel ces OBPs participeraient à la reconnaissance et la libération des molécules odorantes est proposée. Cette thèse fournit des éléments de réponses importants quant à la caractérisation génomique, structurale et fonctionnelle des OBPs de moustiques et peut servir de base de départ pour des recherches expérimentales plus approfondies sur ces aspects

Sequence Analysis and Evolutionary Studies of Reelin Proteins

The reelin gene is conserved across many vertebrate species, including humans. The protein product of this gene plays several important roles in early brain development and regulation of neural network plasticity of a matured brain structure. With an extended structure of 3461 amino acid sequences, consisting of eight reelin repeats, the human reelin sequence stands out as an exceptional model for evolutionary studies. In this study, sequence analysis of the human reelin and its homologues and reelin sequences from 104 other species is described in detail. Interesting sequence conservation patterns of individual repeats have been highlighted. Sequence phylogeny of the reelin sequences indicates a pattern similar to the evolution of the species, thereby serving as a highly conserved family for evolutionary purposes. Multiple sequence alignment of different reelin domain repeats, derived from homologues, suggests specific functions for individual repeats and high sequence conservation across reelin repeats from different organisms, albeit with few unusual domain architectures. A three-dimensional structural model of the full-length human reelin is now available that provides clues on residues at the dimer interface