Population genetics of translational robustness

Recent work has shown that expression level is the main predictor of a gene’s evolutionary rate, and that more highly expressed genes evolve slower. A possible explanation for this observation is selection for proteins which fold properly despite mistranslation, in short selection for translational robustness. Translational robustness leads to the somewhat paradoxical prediction that highly expressed genes are extremely tolerant to missense substitutions but nevertheless evolve very slowly. Here, we study a simple theoretical model of translational robustness that allows us to gain analytic insight into how this paradoxical behavior arises.Comment: 32 pages, 4 figures, Genetics in pres

Frustration in Biomolecules

Biomolecules are the prime information processing elements of living matter. Most of these inanimate systems are polymers that compute their structures and dynamics using as input seemingly random character strings of their sequence, following which they coalesce and perform integrated cellular functions. In large computational systems with a finite interaction-codes, the appearance of conflicting goals is inevitable. Simple conflicting forces can lead to quite complex structures and behaviors, leading to the concept of "frustration" in condensed matter. We present here some basic ideas about frustration in biomolecules and how the frustration concept leads to a better appreciation of many aspects of the architecture of biomolecules, and how structure connects to function. These ideas are simultaneously both seductively simple and perilously subtle to grasp completely. The energy landscape theory of protein folding provides a framework for quantifying frustration in large systems and has been implemented at many levels of description. We first review the notion of frustration from the areas of abstract logic and its uses in simple condensed matter systems. We discuss then how the frustration concept applies specifically to heteropolymers, testing folding landscape theory in computer simulations of protein models and in experimentally accessible systems. Studying the aspects of frustration averaged over many proteins provides ways to infer energy functions useful for reliable structure prediction. We discuss how frustration affects folding, how a large part of the biological functions of proteins are related to subtle local frustration effects and how frustration influences the appearance of metastable states, the nature of binding processes, catalysis and allosteric transitions. We hope to illustrate how Frustration is a fundamental concept in relating function to structural biology.Comment: 97 pages, 30 figure

Regulation of protein homeostasis in neurodegenerative diseases:the role of coding and non-coding genes

Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell's response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding, trafficking and clearance of proteins, all of which act in an orchestrated manner to ensure proteome stability. The protein quality control system is enhanced by stress response pathways, which take action whenever the proteome is challenged by environmental or physiological stress. Aging, however, damages the proteome, and such proteome damage is thought to be associated with aging-related diseases. In this review, we discuss the different cellular processes that define the protein quality control system and focus on their role in protein conformational diseases. We highlight the power of using small organisms to model neurodegenerative diseases and how these models can be exploited to discover genetic modulators of protein aggregation and toxicity. We also link findings from small model organisms to the situation in higher organisms and describe how some of the genetic modifiers discovered in organisms such as worms are functionally conserved throughout evolution. Finally, we demonstrate that the non-coding genome also plays a role in maintaining protein homeostasis. In all, this review highlights the importance of protein and RNA homeostasis in neurodegenerative diseases

Functional and computational identification of a rescue mutation near the active site of an mRNA methyltransferase

RNA-based drugs are an emerging class of therapeutics combining the immense potential of DNA gene-therapy with the absence of genome integration-associated risks. While the synthesis of such molecules is feasible, large scale in vitro production of humanised mRNA remains a biochemical and economical challenge. Human mRNAs possess two post-transcriptional modifcations at their 5′ end: an inverted methylated guanosine and a unique 2′O-methylation on the ribose of the penultimate nucleotide. One strategy to precisely methylate the 2′ oxygen is to use viral mRNA methyltransferases that have evolved to escape the host’s cell immunity response following virus infection. However, these enzymes are ill-adapted to industrial processes and sufer from low turnovers. We have investigated the efects of homologous and orthologous active-site mutations on both stability and transferase activity, and identifed new functional motifs in the interaction network surrounding the catalytic lysine. Our fndings suggest that despite their low catalytic efciency, the active-sites of viral mRNA methyltransferases have low mutational plasticity, while mutations in a defned third shell around the active site have strong efects on folding, stability and activity in the variant enzymes, mostly via network-mediated efects

Structural characterization of f-ALS associated SOD1 mutant aggregates using conformation-specific antibodies

Superoxide dismutase 1 (SOD1) is a metalloenzyme ubiquitously expressed in all cells and acts as an antioxidant enzyme by catalyzing the dismutation of superoxide radicals. Several mutations in the gene encoding SOD1 are linked to the fatal neurodegenerative disease, Amyotrophic lateral sclerosis (ALS). Despite this connection, there is no clear relationship between any quantifiable properties of SOD1 and ALS disease characteristics. One of the key hallmarks of ALS is the formation of SOD1 aggregates in motor neurons. The unfolded SOD1 goes though several post-translational modification steps in vivo, that includes metal binding, disulfide bond formation, and dimerization, to reach its final maturation form which is a stable homodimer. While the mature SOD1 is highly stable, the immature state of SOD1 (E,E SOD1SH) is prone to misfold and aggregate, thereby potentially play a critical role in the disease pathology. Here, we have developed a quantitative dot blot technique to characterise the aggregate structures of several immature SOD1 mutants using four conformation-specific antibodies. These antibodies can only bind to misfolded conformations of SOD1 upon exposure of their binding epitope regions which stays typically buried in the native structure of the protein. The aggregates were prepared in the form of inclusion bodies using the E. coli expression system grown in minimal media. Our results show that different SOD1 mutants exhibit a variable extent of binding to the conformation-specific antibodies, suggesting that SOD1 is displaying mutation-dependent conformational prion-strain behaviour by aggregating into distinct conformations. Hence, the developed quantitative dot blot protocol has provided valuable information on the mechanism of SOD1 aggregation and could potentially be expanded to study aggregate structures of other forms of SOD1 as well as several other proteins that are linked to other neurodegenerative diseases

Evolution favors protein mutational robustness in sufficiently large populations

BACKGROUND: An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual, but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties. RESULTS: Here we use laboratory experiments to demonstrate that evolution favors protein mutational robustness if the evolving population is sufficiently large. We neutrally evolve cytochrome P450 proteins under identical selection pressures and mutation rates in populations of different sizes, and show that proteins from the larger and thus more polymorphic population tend towards higher mutational robustness. Proteins from the larger population also evolve greater stability, a biophysical property that is known to enhance both mutational robustness and evolvability. The excess mutational robustness and stability is well described by existing mathematical theories, and can be quantitatively related to the way that the proteins occupy their neutral network. CONCLUSIONS: Our work is the first experimental demonstration of the general tendency of evolution to favor mutational robustness and protein stability in highly polymorphic populations. We suggest that this phenomenon may contribute to the mutational robustness and evolvability of viruses and bacteria that exist in large populations

Identification And Characterization Of Mediators Of Toxicity Of Aβ Oligomers By Genome Wide Screening In <i>Caenorhabditis Elegans</i>

Soluble oligomers of the amyloid-β (Aβ) protein play a key role in the pathogenesis of Alzheimer’s disease (AD), although the underlying molecular mechanisms are poorly understood. In order to search for proteins involved in the formation and/or toxicity of Aβ oligomers, a transgenic C. elegans model of AD was used in which inducible expression of Aβ oligomers results in a complete paralysis; in these worms a genetic screen following chemical mutagenesis was applied to discover the genes involved in the Aβ-dependent paralysis (forward genetics). This analysis allowed identification of a mutated clone showing a complete lack of paralysis, despite this it not bear mutations in the Aβ coding region, and accumulates Aβ transcript and protein levels comparable to that of the non-mutated strain. This is the first in vivo model in which the expression of Aβ oligomers do not result in any toxic effect. The genome of the mutated worm was then sequenced and compared with that of the control strain to search for altered genes. Two genes, with no known function, were found to bear a stop codon mutation, likely resulting in the translation of an inactive protein. The rest of the mutations were missense mutations. Among them, point mutations were observed in some genes previously correlated with nematode lifespan and ageing. In C. elegans these biological processes are coordinated by the insulin/IGF-1-like signalling (IIS) pathway, which also regulates the response of the organism to toxic aggregated proteins. Thus, the activation of the IIS pathway was investigated in control and mutated worms. As expected, Aβ expression induced the up-regulation of two genes coding for small heat shock proteins (a class of chaperons known to be involved in AD) in the control strain, whereas these genes were actually down-regulated in the mutated strain. Since heat shock proteins are known to bind Aβ oligomers, these chaperons could directly mediate the formation of toxic amyloid species. Moreover, the results of the whole genome sequencing indicate that several proteins could act as potential novel mediators of Aβ toxicity and could open up new insights for research on age-related, neurodegenerative diseases

Physiological aspects underpinning recombinant protein production in Escherichia coli

Many biopharmaceutical projects require the production of recombinant protein in a bacterial host. Conventional procedures used for recombinant protein production (RPP) involve the rapid synthesis of the target protein. This results in the accumulation of unfolded protein, the induction of the heat shock stress response and bacterial growth arrest. More importantly, the target protein accumulates in inclusion bodies and hence is useless to determine its structure. The immediate impact of this is that both the yield and quality of the target protein are compromised. This thesis reports two generically successful approaches that were developed to overcome this series of stress-induced events in Escherichia coli. Both strategies were developed during the production of a cytoplasmic protein and outer membrane lipoproteins using the pET expression system in the bacterial host, E. coli strain BL21(DE3)*. First, the induction protocol was modified to minimise the stress on the host bacterium. This method relies on the induction of very low levels of the T7 RNA polymerase in BL21* and thus the correspondingly slow synthesis of the target protein. Using this approach, growth and productivity of different types of correctly folded target proteins were sustained for at least 70 h. Secondly, mutant hosts that significantly improve recombinant protein production during conventional protocols were isolated. These improved hosts are resistant to IPTG-induced stress and continue to accumulate high levels of the correctly folded target protein. Key to the stress resistance is the presence of mutations that downregulate the synthesis of T7 RNA polymerase. However, different improved hosts were able to enhance the production of different types of target protein, such as those requiring extensive post-translational modification. The potential for isolating a plethora of improved bacterial hosts that are tailored for the production of different types of recombinant protein is discussed in light of the challenges faced by bioindustry. Procedures enabling the isolation of mutant hosts during the production of GFP-tagged and untagged proteins are reported