Quality Control Mechanisms of Molecular Chaperones in the Folding and Degradation of Client Proteins

Molecular chaperones are essential proteins that assist in the folding of substrate ‘client’ proteins to adopt their functionally active three-dimensional structures. The process of protein folding in the cell occurs in a highly concentrated crowded cellular environment among various other macromolecules and amidst various cell stresses which result in issues of aberrant protein folding into toxic species and aggregates. Thus, to counteract these stressors, cells have evolved a complex network of chaperone proteins to maintain protein homeostasis, or proteostasis. Hsp70 is an essential molecular chaperone that acts on clients important for a wide variety of cellular functions. Hsp70 can facilitate refolding of clients to regain their function. However, it can also target client proteins to proteasomal degradation. Turnover of aberrantly folded or aggregation prone proteins such as tau implicates Hsp70 in various pathologies including neurodegenerative diseases. Another class of protein chaperones, termed ‘holdases’, act to delay protein aggregation. The small heat shock proteins (sHSP) systems possess such activity, binding to non-native conformations of clients. sHsps such as Hsp27 and αB crystallin exist as distributions of large oligomeric species that respond dynamically to pH and temperature stresses. Recent studies have demonstrated oligomeric rearrangements occur for sHsps to protect client proteins. A major outstanding question is how do these oligomeric assemblies’ complex structures sense cell stress or protein unfolding or aggregation. In addition to sensing cell stress, sHsps and holdase chaperones are also capable of bridging with the activities of other classes of chaperones, including the Hsp70 chaperone system. Hsp70 functions in concert with a network of co-chaperone proteins which diversify its protein folding capabilities. BAG3 is a nucleotide exchange factor (NEF) that facilitates the exchange of ADP and ATP in Hsp70. In addition, interactions with sHsp family chaperones have emerged, making it a promising target in elucidating the link between these two functionally distinct chaperone systems. The overall theme to my thesis work has been to characterize protein homeostasis achieved through pro-folding and pro-degradation pathways. A major focus of my thesis concerns the ability of Hsp70 to work in concert with the CHIP E3 ubiquitin ligase to target tau for polyubiquitination in a chaperone dependent manner, thus facilitating protein turnover. Another focus has been on a pro-folding function of chaperones, the so-called holdase function, where I have explored the structural rearrangements of the sHsp αB crystallin as well as another multifunctional chaperone, peroxiredoxin, and how these conformational changes and oligomeric rearrangements trigger with external stress and correlate with activation of chaperone activity. This thesis also explores the cooperation between sHsps and Hsp70 to xiii facilitate protein refolding, where I characterize rearrangements that occur in the Hsp27 oligomer distribution modulated by BAG3, and its implications on Hsp70 binding. One of the major techniques utilized in my thesis work is electron microscopy, obtaining structural information of protein complexes, from obtaining low resolution size distributions of sHsp oligomers to pushing resolution of Hsp70 in complex with CHIP beyond quaternary structural information to sub-nanometer resolution of the peroxiredoxin in its active chaperone form in complex with substrate. These studies serve as a foundation for future work on obtaining the structural basis of the decision process where chaperone proteins decide the fate of their client substrates.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144085/1/orvvdom_1.pd

Life’s order, complexity, organization, and its thermodynamic–holistic imperatives

In memoriam Jeffrey S. Wicken (1942–2002)—the evolutionarily minded biochemist, who in the 1970/80s strived for a synthesis of biological and physical theories to fathom the tentative origins of life. Several integrative concepts are worth remembering from Wicken’s legacy. (i) Connecting life’s origins and complex organization to a preexisting physical world demands a thermodynamically sound transition. (ii) Energetic ‘charging’ of the prebiosphere must precede the emergence of biological organization. (iii) Environmental energy gradients are exploited progressively, approaching maximum interactive structure and minimum dissipation. (iv) Dynamic self-assembly of prebiotic organic matter is driven by hydrophobic tension between water and amphiphilic building blocks, such as aggregating peptides from non-polar amino acids and base stacking in nucleic acids. (v) The dynamics of autocatalytic self-organization are facilitated by a multiplicity of weak interactions, such as hydrogen bonding, within and between macromolecular assemblies. (vi) The coevolution of (initially uncoded) proteins and nucleic acids in energy-coupled and metabolically active so-called ‘microspheres’ is more realistic as a kinetic transition model of primal biogenesis than ‘hypercycle replication’ theories for nucleic acid replicators on their own. All these considerations blend well with the current understanding that sunlight UV-induced photo-electronic excitation of colloidal metal sulfide particles appears most suitable as a prebiotic driver of organic synthesis reactions, in tight cooperation with organic, phase-separated, catalytic ‘microspheres’. On the ‘continuist vs. miraculist’ schism described by Iris Fry for origins-of-life considerations (Table 1), Wicken was a fervent early protagonist of holistic ‘continuist’ views and agenda

From protein sequences to 3D-structures and beyond: the example of the UniProt Knowledgebase

With the dramatic increase in the volume of experimental results in every domain of life sciences, assembling pertinent data and combining information from different fields has become a challenge. Information is dispersed over numerous specialized databases and is presented in many different formats. Rapid access to experiment-based information about well-characterized proteins helps predict the function of uncharacterized proteins identified by large-scale sequencing. In this context, universal knowledgebases play essential roles in providing access to data from complementary types of experiments and serving as hubs with cross-references to many specialized databases. This review outlines how the value of experimental data is optimized by combining high-quality protein sequences with complementary experimental results, including information derived from protein 3D-structures, using as an example the UniProt knowledgebase (UniProtKB) and the tools and links provided on its website (http://www.uniprot.org/). It also evokes precautions that are necessary for successful predictions and extrapolations

Systemic studies of RNA binding proteins in stem cell differentiation and pluripotency

What mechanisms govern and maintain cell states during the process of differentiation is a pivotal question in science. What factors govern the commitment of developmental progenitors from pluripotent stem cells is a representative example of this question. Studies of transcriptional, signaling and chromatin regulation have been highly instrumental for elucidating mechanisms pluripotency maintenance. Nevertheless, current knowledge falls short in explaining the exit from pluripotency and its coupling to lineage commitment. It is unclear how pluripotency and differentiation become stabilized in a mutually exclusive manner. Here, I deepen our knowledge concerning post-transcriptional mechanisms in pluripotency-differentiation transition. For this purpose I first characterize by quantitative mass spectrometry the changes that occur in the mRNA bound proteome (RBPome) and identify extensive dynamic rearrangements of the RBPome during early embryonic development, from naive to primed stem cell state and to purified primitive streak progenitors (Chapter I). In parallel I identified developmental post-transcriptional processing landscape and show that the dynamic mRNA binding of the RNA-binding protein TDP-43 is critical in pluripotent stem cells (PSCs) for the choice between self-renewal and differentiation/ pluripotency breakdown (Chapter II). In detail, I discovered that TDP-43 directly regulates an evolutionary conserved switch in alternative polyadenylation (APA) of hundreds of transcripts during early differentiation of mouse and human PSCs. Functional analysis revealed that TDP-43 integrates into pluripotency circuitry by repressing the production of lengthened transcripts of the pluripotency factor SOX2, which is targeted for degradation by miR-21. Furthermore, in pluripotent stem cells TDP-43 also promotes self-renewal by repressing the formation of paraspeckles, membraneless nuclear compartments found only in differentiated cells, by enhancing production of short isoform of the lncRNA NEAT1. Conversely, reduction of TDP-43 during differentiation triggers a short-to-long isoform switch of NEAT1, which polymerizes paraspeckles that in turn recruit TDP-43 and relocalise it away from its other RNA targets. Consistent with this cross-regulation, TDP-43 inhibits differentiation and improves somatic cell reprogramming, while paraspeckles promote early differentiation. These findings reveal how the exit of pluripotency is regulated by a complex posttranscriptional network, which is functionally independent from lineage choices. Apart from its role in the exit of pluripotency, this cross-regulation between paraspeckles and TDP-43 has implications in cancer and neurodegeneration

Bioinformatics

This book is divided into different research areas relevant in Bioinformatics such as biological networks, next generation sequencing, high performance computing, molecular modeling, structural bioinformatics, molecular modeling and intelligent data analysis. Each book section introduces the basic concepts and then explains its application to problems of great relevance, so both novice and expert readers can benefit from the information and research works presented here

Molecular Mechanisms Regulating Chronological Aging and Cell Death in the Toxic Dinoflagellate, Karenia brevis

The toxic dinoflagellate, Karenia brevis, forms nearly annual blooms in the Gulf of Mexico that persist for many months in coastal waters, causing extensive marine animal mortalities and human health impacts. The molecular mechanisms that contribute to cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. The studies presented in this dissertation investigate the existence and involvement of a programmed cell death-like (PCD-like) pathway in the demise of K. brevis cultures following oxidative stress and chronological aging. Firstly, to gain an understanding of the molecular processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out and identified extensive transcriptomic remodeling during the transition into stationary phase indicative of a shift in the metabolic and signaling requirements for survival in a quiescent non-dividing phase. To better understand the connection between the transcriptomic context identified in the microarray study and the presence of a PCO-like pathway in K. brevis, hallmark morphological and biochemical changes (DNA fragmentation, caspase-like activity, and caspase 3-like protein expression) were used to define PCD-like morphological changes following chronological aging and oxidative stress. Targeted in silico bioinformatic mining was used to identify enzymes potentially responsible for the activities observed, as well as the substrates. Finally, K. brevis S-adenosylmethionine synthetase (KbAdoMetS), a putative caspase substrate predicted from the bioinformatics screen, was examined using MALDI-TOF MS to confirm the validity of the bioinformatics approach. Taken together, this work identified that K. brevis contains morphological changes indicative of a caspase-dependent PCD-like pathway and that KbAdoMetS is a caspase 3-like substrate. Finally, we sought to characterize the presence of metacaspases in Karenia brevis, and specifically evaluated the role of metacaspase 1 (KbMC1) during chronological aging and death in culture. Immunocytochemistry, subcellular fractionation, and western blotting results using a custom KbMC1 peptide antibody indicate that KbMC1 may be involved in PCD-like execution through its chloroplastic localization with proposed interactions with the photosynthetic machinery. This study provides the first comprehensive investigation of the molecular processes regulating chronological aging and execution of PCD-like death in a toxic dinoflagellate