Intrinsically disordered proteins link alternative splicing and post-translational modifications to complex cell signaling and regulation

Intrinsically disordered proteins and regions (IDPs and IDRs) lack well-defined tertiary structures, yet carry out various important cellular functions, especially those associated with cell signaling and regulation. In eukaryotes, IDPs and IDRs contain the preferred loci for both alternative splicing (AS) and many post-translational modifications (PTMs). Furthermore, AS and/or PTMs at these loci generally alter the signaling outcomes associated with these IDPs or IDRs, where the functional cooperation of these three features is named the IDP-AS-PTM toolkit. However, the prevalence of such functional modulations remains unknown. Also, the signal-altering mechanisms by which AS, and PTMs modulate function and the extent to which AS and PTMs collaborate in their signaling modulations have not been well defined for particular protein examples. Here we focus on three important signaling and regulatory IDR-containing protein families in humans, namely G-protein coupled receptors (GPCRs), which are transmembrane proteins, the nuclear factors of activated T-cells (NFATs), which are transcription factors (TFs), and the Src family kinases (SFKs), which are signaling enzymes. The goal here is to determine how AS and PTMs individually alter the outcomes of the signaling carried out by the various IDRs and to determine whether AS and PTMs work together to bring about differential cellular responses. We also present data indicating that a wide range of other signaling IDPs or IDRs undergo both AS- and PTM-based modifications, suggesting that they, too, likely take advantage of signal outcome modulations that result from collaboration between these two events. Hence, we propose that the widespread cooperation of IDPs, AS and/or PTMs provides a IDP-AS-PTM toolkit and substantially contributes to the vast complexity of eukaryotic cell signaling systems

Structural and functional characterization of Nucleophosmin domain associated with Acute Myeoloid Leukemia

Nucleophosmin is a shuttleing protein mainly localizated in nucleolus. It is involved in many different cellular processes like ribogenesis, centrosome duplication, apoptotic response. In more then 35% of Acute Myeloyd Leukemic patient with Normal Kariotype, the Nucleophosmin gene is mutated, causing aberrant cytoplasmatic accumulation, imparing DNA binding activity. In this work we describe the structural and functional feuteres of Nucleophosmin DNA binding domain, to better understand its activy both in phisiological and pathologial conditions

What Controls the Controller: Structure and Function Characterizations of Transcription Factor PU.1 Uncover Its Regulatory Mechanism

The ETS family transcription factor PU.1/Spi-1 is a master regulator of the self-renewal of hematopoietic stem cells and their differentiation along both major lymphoid and myeloid branches. PU.1 activity is determined in a dosage-dependent manner as a function of both its expression and real-time regulation at the DNA level. While control of PU.1 expression is well established, the molecular mechanisms of its real-time regulation remain elusive. Our work is focused on discovering a complete regulatory mechanism that governs the molecular interactions of PU.1. Structurally, PU.1 exhibits a classic transcription factor architecture in which intrinsically disordered regions (IDR), consisting of 66% of its primary structure, are tethered to a well-structured DNA binding domain. The transcriptionally active form of PU.1 is a monomer that binds target DNA sites as a 1:1 complex. Our investigations show that IDRs of PU.1 reciprocally control two separate inactive dimeric forms, with and without DNA. At high concentrations, PU.1 forms a non-canonical 2:1 complex at a single DNA specific site. In the absence of DNA, PU.1 also forms a dimer, but it is incompatible with DNA binding. The DNA-free PU.1 dimer is further promoted by phosphomimetic mutants of IDR residues that are phosphorylated in B-lymphocytic activation. These results lead us to postulate a model of real-time PU.1 regulation, unknown in the ETS family, where independent dimeric forms antagonize each other to control the dosage of active PU.1 monomer at its target DNA sites. To demonstrate the biological relevance of our model, cellular assays probing PU.1-specific reporters and native target genes show that PU.1 transactivation exhibits a distinct dose response consistent with negative feedback. In summary, we have established the first model for the general real-time regulation of PU.1 at the DNA/protein level, without the need for recruiting specific binding partners. These novel interactions present potential therapeutic targets for correcting de-regulated PU.1 dosage in hematologic disorders, including leukemia, lymphoma, and myeloma

A computational study of nucleosomal binding and alternative isoforms of human transcription factors

Eukaryotic transcription factors (TFs) are proteins that bind short DNA motifs and regulate gene transcription. Because genomic DNA is organised into nucleosomes via binding histone octamers, TFs compete with histones for binding DNA. Also, the functions of a TF are mainly defined by its domains; therefore, a TF gene can vary the characteristics of its protein product through the expression of alternative isoforms with different domains. However, the mechanisms of TF-nucleosome interactions and the functional importance of alternative TF isoforms are not fully understood. Here, I address these two problems computationally via the integrative analysis of publicly available in vivo human sequencing data. First, I evaluated a novel, gyre-spanning, mode of TF-nucleosome binding proposed recently by another lab based on in vitro evidence. Analysing the nucleosome occupancy and TF binding in the human genome, I found no evidence of such binding and concluded that it must be extremely rare, if at all present. Secondly, I studied the alternative isoforms of human TFs genome-wide. I found that independently of the gene length and the number of exons, TF genes more efficiently sample the set of possible alternative isoforms than non-TF genes, suggesting the particular importance of alternative isoforms for TFs. Also, I found that TF isoforms without a DNA-binding domain (DBD) are produced by almost a third of all human TFs, tend to be tissue-specific and likely reverse the transcription regulation effect of DBD-containing isoforms. Moreover, I demonstrated that the switches of the highest-expressed TF isoforms across human adult tissues may represent a widespread functional mechanism. Finally, I collected a compendium of human TFs with experimentally characterised alternative isoforms which will hopefully serve as a resource for future studies. In summary, my analysis further developed the fundamental knowledge about the TF-nucleosome interactions and the alternative isoforms of TFs in humans.Open Acces

Intrinsically disordered proteins in molecular recognition and structural proteomics

Indiana University-Purdue University Indianapolis (IUPUI)Intrinsically disordered proteins (IDPs) are abundant in nature, being more prevalent in the proteomes of eukaryotes than those of bacteria or archaea. As introduced in Chapter I, these proteins, or portions of these proteins, lack stable equilibrium structures and instead have dynamic conformations that vary over time and population. Despite the lack of preformed structure, IDPs carry out many and varied molecular functions and participate in vital biological pathways. In particular, IDPs play important roles in cellular signaling that is, in part, enabled by the ability of IDPs to mediate molecular recognition. In Chapter II, the role of intrinsic disorder in molecular recognition is examined through two example IDPs: p53 and 14-3-3. The p53 protein uses intrinsically disordered regions at its N- and C-termini to interact with a large number of partners, often using the same residues. The 14-3-3 protein is a structured domain that uses the same binding site to recognize multiple intrinsically disordered partners. Examination of the structural details of these interactions highlights the importance of intrinsic disorder and induced fit in molecular recognition. More generally, many intrinsically disordered regions that mediate interactions share similar features that are identifiable from protein sequence. Chapter IV reviews several models of IDP mediated protein-protein interactions that use completely different parameterizations. Each model has its relative strengths in identifying novel interaction regions, and all suggest that IDP mediated interactions are common in nature. In addition to the biologic importance of IDPs, they are also practically important in the structural study of proteins. The presence of intrinsic disordered regions can inhibit crystallization and solution NMR studies of otherwise well-structured proteins. This problem is compounded in the context of high throughput structure determination. In Chapter III, the effect of IDPs on structure determination by X-ray crystallography is examined. It is found that protein crystals are intolerant of intrinsic disorder by examining existing crystal structures from the PDB. A retrospective analysis of Protein Structure Initiative data indicates that prediction of intrinsic disorder may be useful in the prioritization and improvement of targets for structure determination