Protein Folding Research Articles

Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation.

The intestine plays a key role in metabolism, nutrient and water absorption, and provides both physical and immunological defense against dietary and luminal antigens. The protective mucosal lining in the intestine is a critical component of intestinal barrier that when compromised, can lead to increased permeability, a defining characteristic of inflammatory bowel disease (IBD), among other intestinal diseases. Here, we define a new role for the flavin-containing monooxygenase (FMO) family of enzymes in maintaining a healthy intestinal epithelium. Using Caenorhabditis elegans we measure intestinal barrier function, actin expression, and intestinal damage response. In mice, we utilize an intestine-specific, tamoxifen- inducible knockout model of the mammalian homolog of Cefmo-2 , Fmo5, and assess histology, mucus barrier thickness, and goblet cell physiology. We also treat mice with the ER chaperone Tauroursodeoxycholic acid (TUDCA). In nematodes, we find Cefmo-2 is necessary and sufficient for intestinal barrier function, intestinal actin expression, and is induced by intestinal damage. In mice, we find striking changes to the intestine within two weeks following Fmo5 disruption. Alterations include sex-dependent changes in colon epithelial histology, goblet cell localization, and mucus barrier formation. These changes are significantly more severe in female mice, mirroring differences observed in IBD patients. Furthermore, we find increased protein folding stress in Fmo5 knockout animals and successfully rescue the severe female phenotype with addition of a chemical ER chaperone. Together, our results identify a highly conserved and novel role for Fmo5 in the mammalian intestine and support a key role for Fmo5 in maintenance of ER/protein homeostasis and proper mucus barrier formation.

Atomistic simulations reveal impacts of missense mutations on the structure and function of SynGAP1.

De novo mutations in the synaptic GTPase activating protein (SynGAP) are associated with neurological disorders like intellectual disability, epilepsy, and autism. SynGAP is also implicated in Alzheimer's disease and cancer. Although pathogenic variants are highly penetrant in neurodevelopmental conditions, a substantial number of them are caused by missense mutations that are difficult to diagnose. Hence, in silico mutagenesis was performed for probing the missense effects within the N-terminal region of SynGAP structure. Through extensive molecular dynamics simulations, encompassing three 150-ns replicates for 211 variants, the impact of missense mutations on the protein fold was assessed. The effect of the mutations on the folding stability was also quantitatively assessed using free energy calculations. The mutations were categorized as potentially pathogenic or benign based on their structural impacts. Finally, the study introduces wild-type-SynGAP in complex with RasGTPase at the inner membrane, while considering the potential effects of mutations on these key interactions. This study provides structural perspective to the clinical assessment of SynGAP missense variants and lays the foundation for future structure-based drug discovery.

Endoplasmic reticulum homeostasis in plant–pathogen interactions: new scenarios for an old story

Abstract The endoplasmic reticulum (ER) is a specialized organelle that connects almost all subcellular structures from the plasma membrane to the nucleus. The ER is involved in secretory protein synthesis, folding, and processing. Evidence has emerged that the ER is at the frontier of the battle between plant hosts and pathogens. Its structural and functional homeostasis is crucial for the survival of plant cells. Pathogens secrete effectors to take over normal functions of the ER, while host plants fight back to activate ER stress responses. Exciting advances have been made in studies on host plant–pathogen dynamics during the past decades, namely some new players involved have been recently resolved from both pathogens and hosts. In this review, we summarize advances in identifying structural characteristics of the key pathways and effectors targeting the ER. Newly identified ER-phagy receptors and components downstream of inositol-requiring 1 (IRE1) will be described. Future studies will be envisaged to further our understanding of the missing parts in this dynamic frontier.

Integrated stress response mediates HSP70 to inhibit testosterone synthesis in aging testicular Leydig cells

The integrated stress response (ISR) is implicated in age-related diseases, while the molecular chaperone heat shock protein 70 (HSP70) can facilitate proper protein folding. However, the regulatory mechanism of ISR in insufficient testosterone synthesis of aging Leydig cells (LCs) remains unclear. This study aims to elucidate the regulatory role of ISR in inadequate testosterone synthesis of aging LCs. We observed a positive correlation between testosterone and HSP70 levels, which were found to be decreased in elderly men. ISR was detected in testicular tissue from old mice. The expression of testosterone synthesis related protein and the content of testosterone decreased in testicular tissue of old mice. Conversely, inhibition of the integrated stress response in testicular tissue led to an increase in steroid synthase expression among old mice. Furthermore, inhibiting ISR specifically within aging LCs resulted in enhanced protein translation efficiency and increased expression levels of new HSP70 and steroidogenic acute regulatory protein (StAR). These findings suggest that ISR occurrence within aging LCs affects StAR protein expression through regulation of HSP70-mediated translation, consequently impairing testosterone synthesis.

Mechanical Properties of a Solvated Biomolecule: RGD (1FUV) Peptide.

The mechanical properties of proteins/peptides play an essential role in their functionalities and implications, as well as their structure and dynamic properties. Understanding mechanical properties is pivotal to our knowledge of protein folding and the molecular basis of diverse cellular processes. Herein, we present a computational approach using ab initio quantum mechanical calculations to determine the mechanical properties-such as bulk modulus, shear modulus, Young's modulus, and Poisson's ratio-of a solvated Arg-Gly-Asp (RGD) peptide model. Since this peptide serves as the RGD-directed integrin recognition site and may participate in cellular adhesion, it is considered a promising small peptide for medicinal applications. This successful approach paves the way for investigating larger and more complex biomolecules.

Altered glycosylation in cancer: molecular functions and therapeutic potential

AbstractGlycosylation, a key mode of protein modification in living organisms, is critical in regulating various biological functions by influencing protein folding, transportation, and localization. Changes in glycosylation patterns are a significant feature of cancer, are associated with a range of pathological activities in cancer‐related processes, and serve as critical biomarkers providing new targets for cancer diagnosis and treatment. Glycoproteins like human epidermal growth factor receptor 2 (HER2) for breast cancer, alpha‐fetoprotein (AFP) for liver cancer, carcinoembryonic antigen (CEA) for colon cancer, and prostate‐specific antigen (PSA) for prostate cancer are all tumor biomarkers approved for clinical use. Here, we introduce the diversity of glycosylation structures and newly discovered glycosylation substrate—glycosylated RNA (glycoRNA). This article focuses primarily on tumor metastasis, immune evasion, metabolic reprogramming, aberrant ferroptosis responses, and cellular senescence to illustrate the role of glycosylation in cancer. Additionally, we summarize the clinical applications of protein glycosylation in cancer diagnostics, treatment, and multidrug resistance. We envision a promising future for the clinical applications of protein glycosylation.

Localized ablative immunotherapy enhances antitumor immunity by modulating the transcriptome of tumor-infiltrating Gamma delta T cells

Gamma delta T cells (γδT cells) play crucial roles in the immune response against tumors, yet their functional dynamics under different cancer therapies remain poorly understood. Laser Ablative Immunotherapy (LAIT) is a novel cancer treatment modality combining local photothermal therapy (PTT) and intratumoral injection of an immunostimulant, N-dihydrogalactochitosan (glycated chitosan, GC). LAIT has been shown to induce systemic antitumor immune responses in pre-clinical studies and clinical trials, eradicating both treated local tumors and untreated distant metastases. In this study, we used LAIT to treat breast tumors in a mouse model and investigated the effects of LAIT on tumor-infiltrating γδT cells using single-cell RNA sequencing (scRNAseq). We characterized the γδT cells from tumors in control, PTT, GC, and LAIT (PTT + GC) groups, by identifying six distinct subtypes: activated, cytotoxic, cycling cytotoxic, IFN-enriched, antigen-presenting, and IL17-producing γδT cells. Differential gene expression analysis revealed that LAIT significantly upregulated genes associated with T cell activation, leukocyte adhesion, and interferon signaling in treated tumor tissues while downregulating genes involved in protein folding and stress responses. LAIT also uniquely increased the proportion of IL17-producing γδT cells, which correlated with prolonged survival in breast cancer patients, as analyzed using TCGA data. Furthermore, the transcriptomic profiles of γδT cells in LAIT-treated tumors closely resembled those in immune checkpoint inhibitor (ICI)-treated patients, suggesting potential synergistic effects. Our findings indicate that LAIT modulates the γδT cell transcriptome, enhancing their antitumor capabilities and providing a basis for combining LAIT with ICI therapy to improve cancer treatment outcomes.

An antireductant approach ameliorates misfolded proinsulin-induced hyperglycemia and glucose intolerance in male Akita mice.

Protein folding in the endoplasmic reticulum (ER) requires a high ratio of oxidized to reduced glutathione (GSSG/rGSH). Since the GSSG/rGSH depends on total glutathione (tGSH = GSSG + rGSH) levels, we hypothesized that limiting GSH biosynthesis will ameliorate protein misfolding by enhancing the ER oxidative milieu. As a proof-of-concept, we used DL-buthionine-(S,R)-sulfoximine (BSO) to inhibit GSH biosynthesis in Akita mice, which are prone to proinsulin misfolding. We conducted a 2-week intervention to investigate if BSO was safe and a 6-week intervention to find its effect on glucose intolerance. In both cohorts, male heterozygous Akita (AK) and wild-type (WT) mice were continuously administered 15mM BSO. No adverse effects were observed on body weight, food intake, and water intake in either cohort. Unaltered levels of plasma aspartate and alanine aminotransferases, and cystatin-C, indicate that BSO was safe. BSO-induced decreases in tGSH were tissue-dependent with maximal effects in the kidneys, where it altered the expression of genes associated with GSH biosynthesis, redox status, and proteostasis. BSO treatment decreased random blood glucose levels to 80% and 67%of levels in untreated mice in short-term and long-term cohorts, respectively, and 6-h fasting blood glucose to 82% and 74%of levels in untreated mice, respectively. BSO also improved glucose tolerance by 37% in AK mice in the long-term cohort, without affecting insulin tolerance. Neither glucose tolerance nor insulin tolerance were affected in WT. Data indicate that BSO might treat misfolded proinsulin-induced glucose intolerance. Future studies should investigate the effect of BSO on proinsulin misfolding and if it improves glucose intolerance in individuals with Mutant Insulin Diabetes of Youth.

A Short Review on Introduction and Researches on Anticancerous Activity of Geldanamycin

Geldanamycin (GA) bind heat-shock protein-90 (HSP-90) and destabilize its client proteins including v-Src, Bcr-Abl, RAF-1, Erb-B2, some growth factor receptors and steroid receptors. As a result, several oncoproteins are subjected to ubiquitination and proteasomal destruction by HSP-90 active compounds. HSP-90 active substances can either stop apoptosis from occurring or promote growth arrest, differentiation, and apoptosis depending on the cellular environment. Numerous preclinical models and clinical trials have demonstrated anticancer activity for a number of HSP-90 inhibitors. The well-known HSP-90 inhibitor geldanamycin’s clinical development was hampered by its hepatic toxicity. Geldanamycin at low doses can sensitize Bcr/Abl-expressing leukemia cells to death in the presence of inadequate doxorubicin concentrations by activating caspase. In another example, 17AAG in combination with taxol shows enhanced cytotoxic effects on taxol-resistant Erb-B2 overexpressing breast cancer cells. The benzoquinone ansamycin geldanamycin selectively binds to GRP94 and HSP-90 both in vivo and in vitro. When cells are treated with geldanamycin, HSP-90’s molecular chaperone function is changed. This prevents some cytosolic proteins from maturing, reduces their activity, and/or modifies their stability. On the other hand, nothing is known about GRP94’s function in protein folding or how geldanamycin affects this endoplasmic reticulum (ER) homologue of HSP-90. In this work, we show that geldanamycin is a strong inducer of the cellular stress response in the ER, leading to the transcriptional up-regulation of ER chaperones and production of the gadd153/CHOP transcription factor in a range of cell lines. Here we mention the anticancerous activity of HSP-90 (Heat Shock Protein 90) Inhibitor geldanamycin and some researches in field of anticancerous activity of Geldanamycin.

Mutation/metal deficiency in the "electrostatic loop" enhanced aggregation process in apo/holo SOD1 variants: implications for ALS diseases

Despite the many mechanisms it has created to prevent unfolding and aggregation of proteins, many diseases are caused by abnormal folding of proteins, which are called misfolding diseases. During this process, proteins undergo structural changes and become stable, insoluble beta-sheet aggregates called amyloid fibrils. Mutations/disruptions in metal ion homeostasis in the ALS-associated metalloenzyme superoxide dismutase (SOD1) reduce conformational stability, consistent with the protein aggregation hypothesis for neurodegenerative diseases. However, the exact mechanism of involvement is not well understood. Hence, to understand the role of mutation/ metal deficiency in SOD1 misfolding and aggregation, we investigated the effects of apo/holo SOD1 variants on structural properties using biophysical/experimental techniques. The MD results support the idea that the mutation/metal deficiency can lead to a change in conformation. The increased content of β-sheet structures in apo/holo SOD1 variants can be attributed to the aggregation tendency, which was confirmed by FTIR spectroscopy and dictionary of secondary structure in proteins (DSSP) results. Thermodynamic studies of GdnHCl showed that metal deficiency/mutation/intramolecular S–S reduction together are required to initiate misfolding/aggregation of SOD1. The results showed that apo/holo SOD1 variants under destabilizing conditions induced amyloid aggregates at physiological pH, which were detected by ThT/ANS fluorescence, as well as further confirmation of amyloid/amorphous species by TEM. This study confirms that mutations in the electrostatic loop of SOD1 lead to structural abnormalities, including changes in hydrophobicity, reduced disulfide bonds, and an increased propensity for protein denaturation. This process facilitates the formation of amyloid/amorphous aggregates ALS-associated.

A lightweight visualization tool for protein unfolding by collision detection and elimination

The experiments involving protein denaturation and refolding serve as the foundation for predicting the three-dimensional spatial structures of proteins based on their amino acid sequences. Despite significant progress in protein structure engineering, exemplified by AlphaFold2 and OmegaFold, there remains a gap in understanding the folding pathways of polypeptide chains leading to their final structures. We developed a lightweight tool for protein unfolding visualization called PUV whose graphics design is mainly implemented by OpenGL. PUV leverages principles from molecular biology and physics, and achieves rapid visual dynamics simulation of protein polypeptide chain unfolding through mechanical force and atom-level collision detection and elimination. After a series of experimental validations, we believe that this method can provide essential support for investigating protein folding mechanisms and pathways.

Unraveling the Molecular Pathogenesis of Protein C Deficiency-Associated VTE: Insights from Protein C Mutations C238G and R189W in Thai Patients.

Protein C (PC) deficiency is a well-established risk factor for thromboembolism (TE), commonly manifesting in pediatric patients. This study aimed to elucidate the pathogenic mechanisms of two novel PC mutations, C238G and R189W, identified in Thai children with both venous and arterial TE. The effects of wild-type (WT), C238G, and R189W PC variants were investigated through transient transfection of HEK293T cells. PC secretion levels were measured, and immunofluorescence analysis was performed to assess intracellular localization. ER stress-related gene expression and UPR activation were evaluated. Structural analysis was conducted to explore the significance of the C238 and R189W residue in PC functionality. The C238G mutation led to a severe 95% reduction in PC secretion, while R189W showed a 30% decrease compared with WT. Immunofluorescence revealed that C238G-PC was predominantly retained in the ER, indicating protein misfolding. C238G-expressing cells exhibited significant upregulation of ER stress-related genes and UPR activation. In contrast, R189W resulted in only a modest increase in UPR gene expression, suggesting a less pronounced impact on protein folding and secretion. Structural analysis demonstrated the critical role of the C238 residue in maintaining PC's disulfide bond and overall conformation. This study reveals distinct molecular mechanisms by which the C238G and R189W mutations contribute to PC deficiency and increased thrombotic risk. The findings emphasize the essential role of the C238 residue in preserving PC structure and secretion, enhancing the understanding of PC deficiency-associated TE in pediatric patients.

4-Phenylbutyric acid suppresses psoralen-induced hepatotoxicity by inhibiting ERS and reestablishing mitochondrial fusion-fission balance in mice

Psoralen is a main active molecule of the traditional Chinese herb medicine Fructus Psoraleae. Our previous studies have shown that psoralen induced liver injury through the endoplasmic reticulum stress (ERS) signaling pathways. In this article, we studied whether the ERS inhibitor, 4-phenylbutyrate acid (4-PBA) could inhibit the liver toxicity caused by psoralen, and explored the underlying mechanisms. Mice were given the solvent, 20 mg/kg, 40 mg/kg, 80 mg/kg of psoralen, or 80 mg/kg of psoralen plus 4-PBA for 14 days. We found that 4-PBA significantly reduced the serum LDH and liver tissue MDA level, increased the activities of SOD and CAT, reduced liver weight and coefficient, repaired histopathological damage, and inhibited hepatocytes apoptosis induced by psoralen. RNA-seq transcriptomics found that except for the endoplasmic reticulum, the mitochondria was severely affected by psoralen. And genes involved in mitochondrial fusion, apoptosis, protein folding, and autophagy were found differently expressed in the psoralen group. Further studies found that 4-PBA inhibited the overexpression of GRP78 and CHOP, increased the Bcl-2/Bax ratio, and reduced the expression of Caspase-3. Moreover, 4-PBA reduced the overexpression of mitochondrial fission protein DRP1, increased the expression of fusion proteins Mfn-2 and OPA1, but has no inhibitory effects on autophagy proteins Atg5 or LC3A/B. In conclusion, 4-PBA inhibited ERS and reestablished mitochondrial fusion-fission balance, thereby blocking cell apoptosis, oxidative stress, and mitochondrial dysfunction, thus prevented against psoralen-induced hepatotoxicity.

Solvation free energy in governing equations for DNA hybridization, protein-ligand binding, and protein folding.

This work examines the thermodynamics of model biomolecular interactions using a governing equation that accounts for the participation of bulk water in the equilibria. In the first example, the binding affinities of two DNA duplexes, one of nine and one of 10 base pairs in length, are measured and characterized by isothermal titration calorimetry (ITC) as a function of concentration. The results indicate that the change in solvation free energy that accompanies duplex formation (ΔGS) is large and unfavorable. The duplex with the larger number of G:C pairings yields the largest change in solvation free energy, ΔGS = +460 kcal·mol-1per base pair at 25 °C. A van't Hoff analysis of the data is complicated by the varying degree of intramolecular base stacking within each DNA strand as a function of temperature. A modeling study reveals how the solvation free energy alters the output of a typical ITC experiment and leads to a good, though misleading, fit to the classical equilibrium equation. The same thermodynamic framework is applied to a model protein-ligand interaction, the binding of ribonuclease A with the nucleotide inhibitor 3'-UMP, and to a conformational equilibrium, the change in tertiary structure of α-lactalbumin in molar guanidinium chloride solutions. The ribonuclease study yields a value of ΔGS = +160 kcal·mol-1, whereas the folding equilibrium yields ΔGS ≈ 0, an apparent characteristic of hydrophobic interactions. These examples provide insight on the role of solvation energy in binding equilibria and suggest a pivot in the fundamental application of thermodynamics to solution chemistry.

Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts.

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this "cataract-reversing" phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress-related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.

ITRAQ Based Proteomics Reveals the Potential Mechanism of Placental Injury Induced by Prenatal Stress.

Maternal stress experienced during prenatal development is recognized as a significant risk factor for neurodevelopmental and neuropsychiatric disorders across the offspring's lifespan. The placental barrier serves a crucial function in safeguarding the fetus from detrimental exposures during gestation. However, previous investigations have not yet comprehensively elucidated the extensive connections between prenatal stress and the expression of placental proteins. In this study, we used iTRAQ-based quantitative proteomics to elucidate the placental adaptive mechanisms of pregnant rats in response to fear-induced stress. Our results showed that during pregnancy, exposure to fear-induced stress led to a pathological hypercoagulable state in the mother's body. Placental circulation was also disrupted, significantly reducing placental efficiency and blood oxygen saturation in newborn rats. Proteomic analyses showed that most of the DEPs were annotated to the PI3K-Akt and ECM-receptor interaction signaling pathway. In addition, the expressions of CDC37, HSP90β, AKT, p-AKT and p-mTOR were down-regulated significantly in the placenta. Our results demonstrated that prenatal fear-induced stress led to inhibition of the cellular signal transduction of placental PI3K/AKT/mTOR, which affected biological processes such as rRNA processing, translation, protein folding, protein stability, and oxygen transport in the placenta. These abnormalities in biological functions could potentially damage the barrier function of the placenta and thereby result in abnormal development in the offspring.

Non-cell-autonomous regulation of germline proteostasis by insulin/IGF-1 signaling-induced dietary peptide uptake via PEPT-1

Gametogenesis involves active protein synthesis and is proposed to rely on proteostasis. Our previous work in C. elegans indicates that germline development requires coordinated activities of insulin/IGF-1 signaling (IIS) and HSF-1, the central regulator of the heat shock response. However, the downstream mechanisms were not identified. Here, we show that depletion of HSF-1 from germ cells impairs chaperone gene expression, causing protein degradation and aggregation and, consequently, reduced fecundity and gamete quality. Conversely, reduced IIS confers germ cell resilience to HSF-1 depletion-induced protein folding defects and various proteotoxic stresses. Surprisingly, this effect was not mediated by an enhanced stress response, which underlies longevity in low IIS conditions, but by reduced ribosome biogenesis and translation rate. We found that IIS activates the expression of intestinal peptide transporter PEPT-1 by alleviating its repression by FOXO/DAF-16, allowing dietary proteins to be efficiently incorporated into an amino acid pool that fuels germline protein synthesis. Our data suggest this non-cell-autonomous pathway is critical for proteostasis regulation during gametogenesis.

Characterization of Escherichia coli as a Recombinant Protein Expression Host and its Optimization

Engineered Escherichia coli serves as an efficient platform for the production of a diverse array of recombinant proteins, thereby addressing the pharmaceutical industry's demand for high-quality biological agents. Nevertheless, challenges related to protein folding and the complexities involved in product purification must be acknowledged. This review delineates the advantages and disadvantages of utilizing E. coli as an expression host and further investigates various strategies aimed at optimizing and enhancing the E. coli expression system to elevate the yield, purity, and biological activity of recombinant proteins, ultimately supporting advancements in biopharmaceuticals and other relevant fields.

A Thermostable Heme Protein Fold Adapted for Stereoselective C-H Bond Hydroxylation Using Peroxygenase Activity.

Thermostable protein folds of natural and synthetic origin are highly sought-after templates for biocatalyst generation due to their enhanced stability to elevated temperatures which overcomes one of the major limitations of applying enzymes for synthesis. Cytochrome P450 enzymes (CYPs) are a family of heme-thiolate monooxygenases that catalyse the oxidation of their substrates in a highly stereo- and regio-selective manner. The CYP enzyme (CYP107PQ1) from the thermophilic bacterium Meiothermus ruber binds the norisoprenoid β-ionone and was employed as a scaffold for catalyst design. The I-helix was modified to convert this enzyme from a monooxygenase into a peroxygenase (CYP107PQ1QE), enabling the enantioselective oxidation of β-ionone to (S)-4-hydroxy-β-ionone (94% e.e.). The enzyme was resistant to 20 mM H2O2, 20% (v/v) of organic solvent, supported over 1700 turnovers and was fully functional after incubation at 60 °C for 1 h and 30 °C for 365 days. The reaction was scaled-up to generate multi milligram quantities of the product for characterisation. Overall, we demonstrate that sourcing a CYP protein fold from an extremophile enabled the design of a highly stable enzyme for stereoselective C-H bond activation only using H2O2 as the oxidant, providing a viable strategy for future biocatalyst design.

199 Awardee Talk: Insights into translational regulation through tRNA sequencing, RNA sequencing, and ribosome profiling

Abstract Translation acts as an additional layer of regulation that has an important role in gene expression and function. Due to a phenomenon called codon usage bias, highly expressed genes are thought to be codon-biased to support efficient translation. As more than one codon can code for the same amino acid (synonymous codons), organisms may exhibit preferences for specific codons that facilitate increased expression of important genes due to variations in the availability of corresponding tRNAs. Advances in sequencing technologies have recently permitted the study of the translatome, which refers to the entire population of mRNA associated with ribosomes for protein synthesis and can be investigated through ribosome profiling. This cutting-edge technique allows the identification of actively translated regions, but can also reveal translational pausing events that stem from the presence of SNPs. Our previous research has demonstrated variation in tRNA expression across tissues and different states of health, leading us to consider the connection between tRNA abundance and translational stalling due to SNPs. By coupling ribosome profiling with tRNA sequencing and RNA sequencing, we have investigated all elements of translational machinery to predict translational efficiency, estimate proteome composition, and evaluate tRNA abundance as a source of genetic variation. In this work, we utilized ribosome profiling, tRNAseq, and RNAseq and performed an integrative analysis in bovine tissues (kidney, liver and muscle) as well as murine myoblast cell lines (C2C12). By applying these methods to different bovine tissues, we were able to explore the interplay between tRNA availability and translational stalling events. Moreover, we have identified translationally regulated genes underlying tissue-specific biological processes and found that many upregulated and downregulated genes coincided with high and low translational efficiency respectively. We have also successfully defined stalling sites that depict the regulatory information encoded within the coding sequence of transcripts, which could control translation rate and facilitate proper protein folding. Through the implementation of these next generation sequencing (NGS) methods to cell culture, we were able to evaluate the translatome at various time points (0-min, 30-min, 60-min, and 4-h) post-induction of differentiation. Where most studies have focused on molecular changes between differentiating myoblasts and multinucleated myotubes that are present 7 d after differentiation, we focus on the earliest stages of muscle differentiation that initiate muscle development and therefore kickstart the process of meat production. This work offers an atlas of distinctive stalling sites across bovine tissues and various stages of muscle differentiation, which provides an opportunity to predict codon optimality and understand tissue-specific or time-specific mechanisms of regulating protein synthesis.