A unifying concept that combines the basic features governing self-organization of proteins into complex three-dimensional structures
in vitro and
in vivo is still lacking. Recent experimental ...results and theoretical
in silico modeling studies provide evidence showing that mRNA might contain an additional layer of information, beyond the amino acid sequence, that fine-tunes
in vivo protein folding, which is largely believed to start as a co-translational process. These findings indicate that translation kinetics might direct the co-translational folding pathway and that translational pausing at rare codons might provide a time delay to enable independent and sequential folding of the defined portions of the nascent polypeptide emerging from the ribosome.
Translation of cellular mRNAs via initiation at Internal Ribosome Entry Sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and ...pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.
The study of peptides (synthetic or corresponding to discrete regions of proteins) has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as ...powerful therapeutic agents. However, the functional activity of many short peptides is usually substantially lower than that of their parental proteins. This is (as a rule) due to their diminished structural organization, stability, and solubility often leading to an enhanced propensity for aggregation. Several approaches have emerged to overcome these limitations, which are aimed at imposing structural constraints into the backbone and/or sidechains of the therapeutic peptides (such as molecular stapling, peptide backbone circularization and molecular grafting), therefore enforcing their biologically active conformation and thus improving their solubility, stability, and functional activity. This review provides a short summary of approaches aimed at enhancing the biological activity of short functional peptides with a particular focus on the peptide grafting approach, whereby a functional peptide is inserted into a scaffold molecule. Intra-backbone insertions of short therapeutic peptides into scaffold proteins have been shown to enhance their activity and render them a more stable and biologically active conformation.
In all genomes, most amino acids are encoded by more than one codon. Synonymous codons can modulate protein production and folding, but the mechanism connecting codon usage to protein homeostasis is ...not known. Here we show that synonymous codon variants in the gene encoding gamma-B crystallin, a mammalian eye-lens protein, modulate the rates of translation and cotranslational folding of protein domains monitored in real time by Förster resonance energy transfer and fluorescence-intensity changes. Gamma-B crystallins produced from mRNAs with changed codon bias have the same amino acid sequence but attain different conformations, as indicated by altered in vivo stability and in vitro protease resistance. 2D NMR spectroscopic data suggest that structural differences are associated with different cysteine oxidation states of the purified proteins, providing a link between translation, folding, and the structures of isolated proteins. Thus, synonymous codons provide a secondary code for protein folding in the cell.
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•Quality of protein folding in cells is guided by synonymous codon usage•NMR reveals multiple conformational and oxidation states of synonymous variants•Synonymous codon usage in mRNA alters translation kinetics•Real-time cotranslational folding is guided by synonymous codon usage
Buhr et al. demonstrate how synonymous codon usage guides the real-time kinetics of translation and cotranslational folding of a gamma-B crystallin. Gamma-B crystallins produced from mRNAs with altered codon usage have the same primary sequence but attain different conformations in solution and in the cell.
Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors ...have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNA
binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,β,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNA
to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNA
to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.
Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding ...of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.
Evaluation of: Kimchi-Sarfaty C, Oh JM, Kim IW et al.: A 'silent' polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525-528 (2007) 1 . Individuals carrying silent SNPs in the ...MDR1 gene encoding P-glycoprotein sometimes reveal altered P-glycoprotein pharmacokinetics. There is no rational explanation for why silent SNPs might have such effects, especially when no change in P-glycoprotein mRNA and protein expression levels has been observed. The purpose of this study was to perform careful ex vivo (in cells) analysis of the effects of the three polymorphisms (C1236T, G2677T C3435T) on P-glycoprotein expression and activity. As a result, it has been shown that silent polymorphisms (in particular, C3435T) in MDR1 can alter P-glycoprotein conformation and protein activity/substrate specificity. This study is of immense importance as it demonstrates for the first time that naturally occurring silent SNPs can lead to the synthesis of protein product with the same amino acid sequence but different structural and functional properties. Thus, silent SNPs should no longer be neglected in determining the likelihood of development of various diseases, and should be taken into account in personalized drug treatment and development programs.
Although studies on viral gene expression were essential for the discovery of internal ribosome entry sites (IRESs), it is becoming increasingly clear that IRES activities are present in a ...significant number of cellular mRNAs. Remarkably, many of these IRES elements initiate translation of mRNAs encoding proteins that protect cells from stress (when the translation of the vast majority of cellular mRNAs is significantly impaired). The purpose of this review is to summarize the progress on the discovery and function of cellular IRESs. Recent findings on the structures of these IRESs and specifically regulation of their activity during nutritional stress, differentiation, and mitosis will be discussed.
The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA ...translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits “foamy cell” development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.
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•Protein synthesis during chronic ER stress is independent of eIF2B GEF activity•mRNA translation during chronic ISR is mediated by an eIF3-dependent mechanism•Coordination of transcriptional and translational reprogramming signifies chronic ISR•ER function in chronic ISR relies on PERK-dependent translational reprograming
Guan et al. unravel the mechanism of adaptation to chronic stress that encompasses previously unappreciated remodeling of the translation initiation machinery guided by PERK. These changes in the translation machinery are coordinated with stress-induced transcriptional reprograming and, when disrupted, result in a foamy cell phenotype and cell death.
—In the cell, protein folding begins during protein synthesis/translation and thus is a co-translational process. Co-translational protein folding is tightly linked to translation elongation, which ...is not a uniform process. While there are many reasons for translation non-uniformity, it is generally believed that non-uniform synonymous codon usage is one of the key factors modulating translation elongation rates. Frequent/optimal codons as a rule are translated more rapidly than infrequently used ones and vice versa. Over 30 years ago, it was hypothesized that changes in synonymous codon usage affecting translation elongation rates could impinge on co-translation protein folding and that many synonymous codons are strategically placed within mRNA to ensure a particular translation kinetics facilitating productive step-by-step co-translational folding of proteins. It was suggested that this particular translation kinetics (and, specifically, translation pause sites) may define the window of opportunity for the protein parts to fold locally, particularly at the critical points where folding is far from equilibrium. It was thus hypothesized that synonymous codons may provide a secondary code for protein folding in the cell. Although, mostly accepted now, this hypothesis appeared to be difficult to prove and many convincing results were obtained only relatively recently. Here, I review the progress in the field and explain, why this simple idea appeared to be so challenging to prove.
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