José M. Barral

Non-optimal codon usage affects expression, structure and function of clock protein FRQ

Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes. Codon usage is also implicated in the control of transcription, splicing and RNA structure. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

Function of Trigger Factor and DnaK in Multidomain Protein Folding: Increase in Yield at the Expense of Folding Speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

Real-time observation of trigger factor function on translating ribosomes

The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of ∼10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.

Slowing bacterial translation speed enhances eukaryotic protein folding efficiency.

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.

Silent substitutions predictably alter translation elongation rates and protein folding efficiencies.

Genetic code redundancy allows most amino acids to be encoded by multiple codons that are non-randomly distributed along coding sequences. An accepted theory explaining the biological significance of such non-uniform codon selection is that codons are translated at different speeds. Thus, varying codon placement along a message may confer variable rates of polypeptide emergence from the ribosome, which may influence the capacity to fold toward the native state. Previous studies report conflicting results regarding whether certain codons correlate with particular structural or folding properties of the encoded protein. This is partly due to different criteria traditionally utilized for predicting translation speeds of codons, including their usage frequencies and the concentration of tRNA species capable of decoding them, which do not always correlate. Here, we developed a metric to predict organism-specific relative translation rates of codons based on the availability of tRNA decoding mechanisms: Watson-Crick, non-Watson-Crick or both types of interactions. We determine translation rates of messages by pulse-chase analyses in living Escherichia coli cells and show that sequence engineering based on these concepts predictably modulates translation rates in a manner that is superior to codon usage frequency, which occur during the elongation phase, and significantly impacts folding of the encoded polypeptide. Finally, we demonstrate that sequence harmonization based on expression host tRNA pools, designed to mimic ribosome movement of the original organism, can significantly increase the folding of the encoded polypeptide. These results illuminate how genetic code degeneracy may function to specify properties beyond amino acid encoding, including folding.

Characterization of the inflammatory response during acute and post-acute phases after severe burn.

Severe burn causes a pronounced hypermetabolic response characterized by catabolism and extensive protein wasting. We recently found that this hypermetabolic state is driven by a severe inflammatory response. We characterized in detail the kinetics of serum levels of a panel of cytokines in a rat model, which may serve as reference for the development of therapeutic interventions applicable to humans. Male Sprague-Dawley rats (n = 8) received a full-thickness burn of 60% total body surface area. Serum was harvested 1, 3, 6, 12, 24, 48, 96, and 168 h after burn. Eight serum cytokines commonly used to assess the inflammatory response in humans, such as IL-1&bgr;, IL-6, IL-10, TNF, vascular endothelial growth factor, and monocyte chemotactic protein 1, and the rat-specific cytokines cytokine-induced neutrophil chemoattractant (CINC) 1, CINC-2, and CINC-3 were measured by enzyme-linked immunosorbent assay technique and were compared with controls (n = 4). Statistical analysis was conducted using the t test, with P < 0.05 considered as significantly different. Thermal injury resulted in significantly increased serum levels of IL-1&bgr;, IL-6, IL-10, monocyte chemotactic protein 1, CINC-1, CINC-2, and CINC-3 when compared with the concentrations detected in nonburned rats (P < 0.05). Serum levels of TNF-&agr; and vascular endothelial growth factor in burned rats were not found to be significantly different to controls. Burn causes a profound inflammatory response in rats. Specific cytokines known to increase in humans postburn such as IL-1 &bgr;, IL-6, IL-10, MCP-1, and IL-8 (CINC-1, CINC-2, and CINC-3 in the rat) were also observed in our rat burn model, which now allows us to study new anti-inflammatory treatment options.

Translational tuning optimizes nascent protein folding in cells

Ribosomes help careful protein folding Protein assembly in vitro is useful for studying small molecules but is problematic for studying the assembly of larger, more complex proteins. Kim et al. analyzed the biogenesis of the mutation-prone nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) (see the Perspective by Puglisi). Newly synthesized polypeptides emerged relatively slowly from the ribosome and folded through a modulated pathway that ensured correct protein folding. Some parts of the protein chain folded immediately upon synthesis, whereas other segments did so more slowly. It appears that acquiring the correct conformation for this complex protein is partly guided by the ribosome itself. Science, this issue p. 444; see also p. 399 De novo CFTR folding is monitored at critical stages of synthesis. [Also see Perspective by Puglisi] In cells, biosynthetic machinery coordinates protein synthesis and folding to optimize efficiency and minimize off-pathway outcomes. However, it has been difficult to delineate experimentally the mechanisms responsible. Using fluorescence resonance energy transfer, we studied cotranslational folding of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator. During synthesis, folding occurred discretely via sequential compaction of N-terminal, α-helical, and α/β-core subdomains. Moreover, the timing of these events was critical; premature α-subdomain folding prevented subsequent core formation. This process was facilitated by modulating intrinsic folding propensity in three distinct ways: delaying α-subdomain compaction, facilitating β-strand intercalation, and optimizing translation kinetics via codon usage. Thus, de novo folding is translationally tuned by an integrated cellular response that shapes the cotranslational folding landscape at critical stages of synthesis.

Identification of Nascent Chain Interaction Sites on Trigger Factor

The role of ribosome-binding molecular chaperones in protein folding is not yet well understood. Trigger factor (TF) is the first chaperone to interact with nascent polypeptides as they emerge from the bacterial ribosome. It binds to the ribosome as a monomer but forms dimers in free solution. Based on recent crystal structures, TF has an elongated shape, with the peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and the N-terminal ribosome binding domain positioned at opposite ends of the molecule and the C-terminal domain, which forms two arms, positioned in between. By using site specifically labeled TF proteins, we have demonstrated that all three domains of TF interact with nascent chains during translation. Interactions with the PPIase domain were length-dependent but independent of PPIase activity. Interestingly, with free TF, these same sites were found to be involved in forming the dimer interface, suggesting that dimerization partially occludes TF-nascent chain binding sites. Our data indicate the existence of two regions on TF along which nascent chains can interact, the NC-domains as the main site and the PPIase domain as an auxiliary site.

Calcium and ER stress mediate hepatic apoptosis after burn injury

A hallmark of the disease state following severe burn injury is decreased liver function, which results in gross metabolic derangements that compromise patient survival. The underlying mechanisms leading to hepatocyte dysfunction after burn are essentially unknown. The aim of the present study was to determine the underlying mechanisms leading to hepatocyte dysfunction and apoptosis after burn. Rats were randomized to either control (no burn) or burn (60% total body surface area burn) and sacrificed at various time-points. Liver was either perfused to isolate primary rat hepatocytes, which were used for in vitro calcium imaging, or liver was harvested and processed for immunohistology, transmission electron microscopy, mitochondrial isolation, mass spectroscopy or Western blotting to determine the hepatic response to burn injury in vivo. We found that thermal injury leads to severely depleted endoplasmic reticulum (ER) calcium stores and consequent elevated cytosolic calcium concentrations in primary hepatocytes in vitro. Burn-induced ER calcium depletion caused depressed hepatocyte responsiveness to signalling molecules that regulate hepatic homeostasis, such as vasopressin and the purinergic agonist ATP. In vivo, thermal injury resulted in activation of the ER stress response and major alterations in mitochondrial structure and function - effects which may be mediated by increased calcium release by inositol 1,4,5-trisphosphate receptors. Our results reveal that thermal injury leads to dramatic hepatic disturbances in calcium homeostasis and resultant ER stress leading to mitochondrial abnormalities contributing to hepatic dysfunction and apoptosis after burn injury.

Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein

Background: Ubiquilin-1 may contribute to the development of Alzheimer disease; however, the mechanisms are unclear. Results: Ubiquilin-1 functions as a molecular chaperone, binding to and preventing the aggregation of amyloid precursor protein. Conclusion: The molecular chaperone function of ubiquilin-1 contributes to amyloid precursor protein biosynthesis and processing. Significance: Decreased quality control of amyloid precursor protein by ubiquilin-1 may contribute to late onset Alzheimer disease pathogenesis. Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.