Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

Farnesylation is an important post-translational modification catalyzed by farnesyltransferase (FTase). Until recently it was believed that a C-terminal CaaX motif is required for farnesylation, but recent experiments have revealed larger substrate diversity. In this study, we propose a general structural modeling scheme to account for peptide binding specificity and recapitulate the experimentally derived selectivity profile of FTase in vitro. In addition to highly accurate recovery of known FTase targets, we also identify a range of novel potential targets in the human genome, including a new substrate class with an acidic C-terminal residue (CxxD/E). In vitro experiments verified farnesylation of 26/29 tested peptides, including both novel human targets, as well as peptides predicted to tightly bind FTase. This study extends the putative range of biological farnesylation substrates. Moreover, it suggests that the ability of a peptide to bind FTase is a main determinant for the farnesylation reaction. Finally, simple adaptation of our approach can contribute to more accurate and complete elucidation of peptide-mediated interactions and modifications in the cell

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

BACKGROUND: In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations. METHODOLOGY/PRINCIPAL FINDINGS: T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4(+) T cells, ii) CXCR3 in CD8(+) T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vdelta2gamma9 T cells, and upregulated CXCR4 expression in TCR Vdelta2gamma9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4(+) T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8(+) T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vdelta2gamma9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T(FH)) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T(FH) and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T(FH) cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, beta-arrestin and SHP2 was modulated by sHLA-G treatment. CONCLUSIONS/SIGNIFICANCE: Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions

Reconstruction of Genome-Scale Active Metabolic Networks for 69 Human Cell Types and 16 Cancer Types Using INIT

Development of high throughput analytical methods has given physicians the potential access to extensive and patient-specific data sets, such as gene sequences, gene expression profiles or metabolite footprints. This opens for a new approach in health care, which is both personalized and based on system-level analysis. Genome-scale metabolic networks provide a mechanistic description of the relationships between different genes, which is valuable for the analysis and interpretation of large experimental data-sets. Here we describe the generation of genome-scale active metabolic networks for 69 different cell types and 16 cancer types using the INIT (Integrative Network Inference for Tissues) algorithm. The INIT algorithm uses cell type specific information about protein abundances contained in the Human Proteome Atlas as the main source of evidence. The generated models constitute the first step towards establishing a Human Metabolic Atlas, which will be a comprehensive description (accessible online) of the metabolism of different human cell types, and will allow for tissue-level and organism-level simulations in order to achieve a better understanding of complex diseases. A comparative analysis between the active metabolic networks of cancer types and healthy cell types allowed for identification of cancer-specific metabolic features that constitute generic potential drug targets for cancer treatment

Expression and prognostic impact of the protein tyrosine phosphatases PRL-1, PRL-2, and PRL-3 in breast cancer

The aim of this study was to investigate the expression of the protein tyrosine phosphatases (PTP) PRL-1, PRL-2, and PRL-3 in human breast cancer and to evaluate its clinical and prognostic significance. PRL-PTP mRNA expression was examined in malignant (n=7) and nonmalignant (n=7) cryoconserved breast tissue samples as well as in eight breast cancer cell lines by RT–PCR. Furthermore, protein expression of PRL-3 was analysed semiquantitatively by immunohistochemistry in ductal breast carcinoma in situ (n=135) and invasive breast cancer (n=147) by use of tissue microarray technology (TMA). In 24 lymph node-positive patients we selected the corresponding lymph node metastases for analysis of PRL-3 expression, and a validation set (n=99) of invasive breast cancer samples was examined. Staining results were correlated with clinicopathological parameters and long-term follow-up. PRL-3 mRNA expression was significantly higher in malignant compared to benign breast tissue. For PRL-1 and PRL-2 expression no significant differences were observed. Staining of TMAs showed PRL-3 expression in 85.9% ductal carcinoma in situ and 75.5% invasive breast carcinomas. Analysis of survival parameters revealed a shorter disease-free survival (DFS) in patients with PRL-3-positive carcinomas, and in particular a significantly shorter DFS in nodal-positive patients with PRL-3 overexpressing tumours as compared to PRL-3-negative breast carcinomas (66±7 months (95% CI, 52–80) vs 97±9 months (95% CI, 79–115); P=0.032). Moreover, we found a more frequent expression of PRL-3 in lymph node metastases as compared to the primary tumours (91.7 vs 66.7%; P=0.033). Our results suggest that PRL-3 might serve as a novel prognostic factor in breast cancer, which may help to predict an adverse disease outcome

Novel Allosteric Sites on Ras for Lead Generation

Aberrant Ras activity is a hallmark of diverse cancers and developmental diseases. Unfortunately, conventional efforts to develop effective small molecule Ras inhibitors have met with limited success. We have developed a novel multi-level computational approach to discover potential inhibitors of previously uncharacterized allosteric sites. Our approach couples bioinformatics analysis, advanced molecular simulations, ensemble docking and initial experimental testing of potential inhibitors. Molecular dynamics simulation highlighted conserved allosteric coupling of the nucleotide-binding switch region with distal regions, including loop 7 and helix 5. Bioinformatics methods identified novel transient small molecule binding pockets close to these regions and in the vicinity of the conformationally responsive switch region. Candidate binders for these pockets were selected through ensemble docking of ZINC and NCI compound libraries. Finally, cell-based assays confirmed our hypothesis that the chosen binders can inhibit the downstream signaling activity of Ras. We thus propose that the predicted allosteric sites are viable targets for the development and optimization of new drugs

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field