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Regulation of gene expression occurs at several levels in a cell. While control of transcription is often viewed as the main source of regulation, it is now clear that post-transcriptional processes are essential to fine-tune protein availability. The presence of AU-rich elements (ARE) in the 3’ untranslated region (3’UTR) of many important mRNAs exemplifies one such process. AREs alter the mRNA translation or degradation status by recruiting ARE-binding proteins (ARE-BP). ARE-BPs of the TTP/TIS11 family bind to their cognate ARE-RNAs using their conserved tandem zinc-finger domain and induce rapid decay of their targets. This allows for proper regulation of cell proliferation, cell death and inflammation. In this regard, TTP/TIS11 are main regulators of gene expression, and as such are put under strict transcriptional, post-transcriptional as well as several layers of post-translational control.In this work, we aimed at elucidating the degradation mechanisms affecting TTP/TIS11. Using Drosophila as a model, we found that dTIS11 protein turnover is rapid due to continuous degradation by the proteasome. However, proteasomal recognition did not require ubiquitination of dTIS11 as non-ubiquitinable mutants were efficiently degraded by the proteasome. In addition, dTIS11 was digested by the 20S proteasome that lacks ubiquitin-recognition domains. Our results further indicate that intrinsically disordered regions (IDR) in dTIS11 may be responsible for proteasomal recognition. In fact, dTIS11 is predicted as disordered and possesses the main characteristics of intrinsically disordered proteins (IDP). We also identified dTIS11 N- and C-terminal domains as functional signals for degradation, potentially due to their destructuration. This ubiquitination-independent, disorder-dependent degradation process is conserved throughout evolution as dTIS11 mammalian counterpart, TTP, undergoes the same degradation by default pathway. In addition, we established that phosphorylatio, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

info:eu-repo/semantics/published

info:eu-repo/semantics/published

Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined "fuzziness'', often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-pi interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation-pi contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-pi hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-pi model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-pi contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs.

Protein function and dynamics are closely related; however, accurate dynamics information is difficult to obtain. Here based on a carefully assembled data set derived from experimental data for proteins in solution, we quantify backbone dynamics properties on the amino-acid level and develop DynaMine--a fast, high-quality predictor of protein backbone dynamics. DynaMine uses only protein sequence information as input and shows great potential in distinguishing regions of different structural organization, such as folded domains, disordered linkers, molten globules and pre-structured binding motifs of different sizes. It also identifies disordered regions within proteins with an accuracy comparable to the most sophisticated existing predictors, without depending on prior disorder knowledge or three-dimensional structural information. DynaMine provides molecular biologists with an important new method that grasps the dynamical characteristics of any protein of interest, as we show here for human p53 and E1A from human adenovirus 5., info:eu-repo/semantics/published

info:eu-repo/semantics/published

info:eu-repo/semantics/published