Ravi Ramesh Pathak

Pathological Unfoldomics of Uncontrolled Chaos: Intrinsically Disordered Proteins and Human Diseases

Many biologically important proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro as a whole or in part.1–5 These intrinsically disordered proteins (IDPs), or intrinsically disordered protein regions (IDPRs) of hybrid proteins possessing both structured and disordered domains, do not have unique well-defined 3D structures, existing instead as collapsed or extended dynamically mobile conformational ensembles. Therefore, natural proteins can be found in one of three major protein forms: functional and folded, nonfunctional and misfolded, or functional and intrinsically disordered. Although IDPs and IDPRs are highly dynamic, their structures can be described reasonably well by a rather limited number of lower-energy conformations.6,7 The structural plasticity and conformational adaptability of IDPs/IDPRs and their intrinsic lack of rigid structure leads to a number of exceptional functional advantages, providing them with unique capabilities to act in functional modes not achievable by ordered proteins.5 As a result, intrinsic disorder is a common feature of proteins involved in signaling, regulation, and recognition, and IDPs/IDPRs play diverse roles in modulation and control of their binding partners’ functions and in promoting the assembly of supramolecular complexes. The biological actions of IDPs/IDPRs, which frequently serve as major regulators of their binding partners, are controlled by extensive posttranslational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, and sumoylation,5 and by alternative splicing.8 In fact, many IDPs/IDPRs are known to contain multiple functional elements that contribute to their ability to be involved in interaction with, regulation of, and control by multiple structurally unrelated partners.9 Given the existence of multiple functions in a single disordered protein, and given that each functional element is typically relatively short, alternative splicing could readily generate sets of protein isoforms with highly diverse regulatory elements.8 The complexity of the disorder-based interactomes is further increased by the capacity of a single IDPR to bind to multiple partners, gaining very different structures in the bound state.10 IDPs can form highly stable complexes or be involved in signaling interactions where they undergo constant “bound–unbound” transitions, thus acting as dynamic and sensitive “on–off” switches. The ability of these proteins to return to highly flexible conformations after the completion of a particular function, and their predisposition to adopt different conformations depending on their environment, are unique physiological properties of IDPs that allow them to exert different functions in different cellular contexts according to a specific conformational state.5 Although the field of protein disorder has started from careful analysis of a very limited number of biologically active proteins without unique structures (which, for a long time, were taken as rare exceptions from the general “one sequence–one unique structure–one unique function” paradigm),1–4 applications of various disorder predictors to different proteomes revealed that IDPs are highly abundant in nature,11–16 and the overall amount of disorder in proteins increases from bacteria to archaea to eukaryota, with over half of all eukaryotic proteins predicted to contain extended IDPRs.11,12,15–17 One explanation for this trend is a change in the cellular requirements for certain protein functions, particularly cellular signaling. In support of this hypothesis, an analysis of a eukaryotic signal protein database indicated that the majority of known signal transduction proteins were predicted to contain significant regions of disorder.18 A detailed study focused on the intricate mechanisms of IDP regulation inside the cell was recently conducted by Gsponer et al.19 These authors grouped all the Saccharomyces cerevisiae proteins into three classes according to their predicted disorder propensities and evaluated the correlations between intrinsic disorder and the various regulation steps of protein synthesis and degradation.19 Although the transcriptional rates of mRNAs encoding IDPs and ordered proteins were comparable, IDP-encoding transcripts were generally less abundant than transcripts encoding ordered proteins because of increased decay rates of IDP mRNAs.19 Also, IDPs were found to be less abundant than ordered proteins because of lower rates of protein synthesis and shorter protein half-lives.19 Curiously, IDPs were shown to be substrates of twice as many kinases as ordered proteins. Furthermore, the vast majority of kinases whose substrates were IDPs were either regulated in a cell-cycle-dependent manner or activated upon exposure to specific stimuli or stress.19 Similar regulation trends were also found in proteomes of Schizosaccharomyces pombe and Homo sapiens,19 suggesting that both unicellular and multicellular organisms use evolutionarily conserved mechanisms to regulate the availability of their IDPs. This tight regulation is directly related to the major roles of IDPs/IDPRs in signaling, where it is crucial for a given protein to be available in appropriate amounts and not to be present longer than needed.19 It was also pointed out5 that although the abundance of many IDPs may be closely regulated, some disordered proteins could be present in cells in large amounts or/and for long periods of time, either due to specific PTMs or via interactions with other factors. These events could promote changes in cellular localization of IDPs or protect them from degradation.3,20–23 Taken together, these data highlight that the chaos seemingly associated with highly flexible and promiscuous IDPs/IDPRs is under tight control.24

Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional Versatility

IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTENs protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.

Loss of phosphatase and tensin homolog (PTEN) induces leptin-mediated leptin gene expression: feed-forward loop operating in the lung.

Background: Leptin expression is induced in lung diseases and lung cancer, but the mechanism of leptin gene expression remains elusive. Results: Leptin mediates leptin and leptin receptor expression, setting up a feed-forward loop. Conclusion: DNA elements and intracellular signals activating leptin gene expression were identified. Significance: Mechanism of leptin/leptin receptor gene regulation will aid in targeting leptin signaling in lung pathologies. Elevated levels of systemic and pulmonary leptin are associated with diseases related to lung injury and lung cancer. However, the role of leptin in lung biology and pathology, including the mechanism of leptin gene expression in the pathogenesis of lung diseases, including lung cancer, remains elusive. Here, using conditional deletion of tumor suppressor gene Pten in the lung epithelium in vivo in transgenic mice and human PTEN-null lung epithelial cells, we identify the leptin-driven feed-forward signaling loop in the lung epithelial cells. Leptin-mediated leptin/leptin-receptor gene expression likely amplifies leptin signaling that may contribute to the pathogenesis and severity of lung diseases, resulting in poor clinical outcomes. Loss of Pten in the lung epithelial cells in vivo activated adipokine signaling and induced leptin synthesis as ascertained by genome-wide mRNA profiling and pathway analysis. Leptin gene transcription was mediated by binding of transcription factors NRF-1 and CCAAT/enhancer-binding protein δ (C/EBP) to the proximal promoter regions and STAT3 to the distal promoter regions as revealed by leptin promoter-mutation, chromatin immunoprecipitation, and gain- and loss-of-function studies in lung epithelial cells. Leptin treatment induced expression of the leptin/leptin receptor in the lung epithelial cells via activation of MEK/ERK, PI3K/AKT/mammalian target of rapamycin (mTOR), and JAK2/STAT3 signaling pathways. Expression of constitutively active MEK-1, AKT, and STAT3 proteins increased expression, and treatment with MEK, PI3K, AKT, and mTOR inhibitors decreased LEP expression, indicating that leptin via MAPK/ERK1/2, PI3K/AKT/mTOR, and JAK2/STAT3 pathways, in turn, further induces its own gene expression. Thus, targeted inhibition of the leptin-mediated feed-forward loop provides a novel rationale for pharmacotherapy of disease associated with lung injury and remodeling, including lung cancer.

Integrating omics technologies to study pulmonary physiology and pathology at the systems level.

Assimilation and integration of “omics” technologies, including genomics, epigenomics, proteomics, and metabolomics has readily altered the landscape of medical research in the last decade. The vast and complex nature of omics data can only be interpreted by linking molecular information at the organismic level, forming the foundation of systems biology. Research in pulmonary biology/medicine has necessitated integration of omics, network, systems and computational biology data to differentially diagnose, interpret, and prognosticate pulmonary diseases, facilitating improvement in therapy and treatment modalities. This review describes how to leverage this emerging technology in understanding pulmonary diseases at the systems level -called a “systomic” approach. Considering the operational wholeness of cellular and organ systems, diseased genome, proteome, and the metabolome needs to be conceptualized at the systems level to understand disease pathogenesis and progression. Currently available omics technology and resources require a certain degree of training and proficiency in addition to dedicated hardware and applications, making them relatively less user friendly for the pulmonary biologist and clinicians. Herein, we discuss the various strategies, computational tools and approaches required to study pulmonary diseases at the systems level for biomedical scientists and clinical researchers.

Structural pliability adjacent to the kinase domain highlights contribution of FAK1 IDRs to cytoskeletal remodeling

Therapeutic protein kinase inhibitors are designed on the basis of kinase structures. Here, we define intrinsically disordered regions (IDRs) in structurally hybrid kinases. We reveal that 65% of kinases have an IDR adjacent to their kinase domain (KD). These IDRs are evolutionarily more conserved than IDRs distant to KDs. Strikingly, 36 kinases have adjacent IDRs extending into their KDs, defining a unique structural and functional subset of the kinome. Functional network analysis of this subset of the kinome uncovered FAK1 as topologically the most connected hub kinase. We identify that KD-flanking IDR of FAK1 is more conserved and undergoes more post-translational modifications than other IDRs. It preferentially interacts with proteins regulating scaffolding and kinase activity, which contribute to cytoskeletal remodeling. In summary, spatially and evolutionarily conserved IDRs in kinases may influence their functions, which can be exploited for targeted therapies in diseases including those that involve aberrant cytoskeletal remodeling.

Data on evolution of intrinsically disordered regions of the human kinome and contribution of FAK1 IDRs to cytoskeletal remodeling

We present data on the evolution of intrinsically disordered regions (IDRs) taking into account the entire human protein kinome. The evolutionary data of the IDRs with respect to the kinase domains (KDs) and kinases as a whole protein (WP) are reported. Further, we have reported its post translational modifications of FAK1 IDRs and their contribution to the cytoskeletal remodeling. We also report the data to build a protein-protein interaction (PPI) network of primary and secondary FAK1-interacting hybrid proteins. Detailed analysis of the data and its effect on FAK1-related functions have been described in “Structural pliability adjacent to the kinase domain highlights contribution of FAK1 IDRs to cytoskeletal remodeling” (Kathiriya et. al., 2016) [1].

EDITORIAL : Imaging Mass Spectrometry: Advancing systems approaches for biological sciences

The impact of technology in modern science is unequivocal. As scientific thought and design moves from traditional reductionism towards holism,...

Protein Structure and Function: Methods for Prediction and Analysis

Protein structure and functions that are associated with it have been studied extensively. Traditional methods of studying protein and structure have largely relied on biochemical and biophysical characterization methods. However, these methods rely on the assumption that proteins are largely structured and adopt energetically stable three-dimensional conformations with minimum free energy. The discovery of naturally unfolded proteins or disordered proteins that are characterized by lack of stable tertiary structure paved the way to study hitherto unexplained facets of protein structure and function. There are a number of computational methods that exploit protein sequence information to predict whether a protein is disordered. These are complemented by a number of other tools that allow users to predict protein function based on the occurrence of post translational modifications, short linear motifs and other disorder associated regions driving functional plasticity. These easy to apply and interpret tools are presented in the form of a user-friendly workflow for bench-scientists in the current communication.

CancerDR: An overview of the online database for cancer drug resistance

U.S. President Richard Nixon signed the National Cancer Act of 1971 that galvanized efforts aimed at seeking a cure for the disease and better understand the biological events that lead to it.It has been 40 years since the eventful declaration by the Nixon Administration, however despite significant progress in terms of understanding the mechanisms of the disease and some notable treatment successes at early onset of the disease; cancer continues to be a major cause of death[1]. Chemotherapy has been the mainstays of treatment for cancer patients, which is fraught with numerous side effects and is associated with poor quality of life for patients. With an increasing understanding of the molecular mechanisms of cancer, targeted therapies aimed at disrupting signaling pathways and Journal of Postdoctoral Research www.postdocjournal.com