Protein Elongation Research Articles

Non-thermal shearing effect on gluten conformation for plant-based anisotropic structures

Wheat gluten manipulation to obtain fibrillar structures is a promising approach for elaborating meat analogs on large or small scales. Alternatively to extrusion, mild methods can save energy and avoid sensory attributes/protein degradation. This study aimed to evaluate low-temperature shearing to create gluten-based fibers, assessing the material structure as a function of processing intensity. The effect of the non-thermal shearing length (up to 10 min) and added mechanical energy (up to 565.5 J/g) applied on a highly hydrated wheat gluten-based matrix was investigated in terms of density, exudation, secondary protein structure (FTIR), protein network attributes (confocal laser scanning microscopy), elongation attributes, morphology (SEM), and anisotropicity of cooked matrices. The progression of the shearing process was associated with the development of β-sheet secondary structures (26 % to 50 % predominancy), higher elongation attributes, raw matrix density, and anisotropicity of cooked matrices. Higher protein vessel length and width were associated with the formation and stacking of β-sheets. Exudation, cooked matrix density, and endpoint rate were associated with higher α-helices content (lowered from 39 % to 11 % of predominancy) and lower shearing-added energies. A principal component analysis of the entire dataset confirmed these observations, and the morphology revealed the evolution of the matrix organization during the shearing process. These results underscore the potential of mild-temperature shearing of a highly hydrated gluten-enriched matrix to alter the protein conformation, opening possibilities for controlling fiber structure development, valid for new foods such as meat analogs.

Homo-harringtonine (HHT) is highly effective against SARS-CoV-2-A potential first-line defense in future coronavirus epidemics

Abstract As COVID-19 is the third coronavirus epidemic in this century, it would be desirable to have a treatment scheme in anticipation of a fourth one. We here present a scheme that could clear SARS-CoV-2 in 2-4 days post infection from the upper respiratory tract (URT), where the virions are initially concentrated. The scheme, applicable in large scale by nasal spray, is based on Homo-harringtonine (HHT) that has been approved for treating other diseases. HHT blocked protein elongation and repressed in vitro replication of all 4 coronaviruses (including SARS-CoV-2) tested at the nano-molar concentration, demonstrating its potential of broad effectiveness against coronaviruses. In animal models, HHT cleared SARS-CoV-2 in all treated mice in 3 days by daily nasal dripping of a small dose (40 ug). In December 2022, HHT was administered to 26 cancer patients by nebulization at 1 mg/day. On average, the viral load in the URT was reduced by 3/4 six hours after the nebulization. In the wavelet of May 2023, 11 patients without other medical conditions were administered HHT by repeated liquid nasal spray at the low, total daily-dose of 0.2 mg. Ten of the 11 patients were cleared of the virus in 2-4 days. In comparison, in large-cohort studies of participants in China during the same wave, most patients need 7-9 to turn negative. No adverse effects were detected in any patient in the two clinical trials. A short review of drugs approved for treating COVID-19 shows the many advantages of HHT. With continual development, it could become a first-line defense at the onset of future coronavirus epidemics.

Transcriptomic and Proteomic Integration Reveals Key Tapping-Responsive Factors for Natural Rubber Biosynthesis in the Rubber Tree Hevea brasiliensis

Natural rubber is a crucial industrial material, and it is primarily harvested from the latex of the rubber tree Hevea brasiliensis by tapping the tree trunk. During the regular tapping process, mechanical damage seriously affects latex reproduction and rubber yield, but the molecular mechanisms on tapping stimulation remain unclear. In this study, we firstly determined the changed physiological markers on latex regeneration, overall latex yield, and latex flow time during the tapping process. Then, we combined proteomics and transcriptomics analyses of latex during tapping and identified 3940 differentially expressed genes (DEGs) and 193 differentially expressed proteins (DEPs). Among them, 773 DEGs and 120 DEPs displayed a persistent upregulation trend upon tapping. It is interesting that, in the detected transcription factors, basic helix-loop-helix (bHLH) family members occupied the highest proportion among all DEGs, and this trend was similarly observed in DEPs. Notably, 48 genes and 34 proteins related to natural rubber biosynthesis were identified, and most members of small rubber particle protein (SRPP) and rubber elongation factor (REF) showed a positive response to tapping stimulation. Among them, SRPP6 and REF5 showed significant and sustained upregulation at the gene and protein levels following tapping, indicating their pivotal roles for post-tapping rubber biosynthesis. Our results deepen the comprehension of the regulation mechanism underlying tapping and provide candidate genes and proteins for improving latex production in the Hevea rubber tree in future.

Branched-chain amino acids alleviate NAFLD via inhibiting de novo lipogenesis and activating fatty acid β-oxidation in laying hens

The adverse metabolic impacts of branched-chain amino acids (BCAA) have been elucidated are mediated by isoleucine and valine. Dietary restriction of isoleucine promotes metabolic health and increases lifespan. However, a high protein diet enriched in BCAA is presently the most useful therapeutic strategy for nonalcoholic fatty liver disease (NAFLD), yet, its underlying mechanism remains largely unknown. Fatty liver hemorrhagic syndrome (FLHS), a specialized laying hen NAFLD model, can spontaneously develop fatty liver and hepatic steatosis under a high-energy and high-protein dietary background that the pathogenesis of FLHS is similar to human NAFLD. The mechanism underlying dietary BCAA control of NAFLD development in laying hens remains unclear. Herein, we demonstrate that dietary supplementation with 67 % High BCAA has unique mitigative impacts on NAFLD in laying hens. A High BCAA diet alleviates NAFLD, by inhibiting the tryptophan-ILA-AHR axis and MAPK9-mediated de novo lipogenesis (DNL), promoting ketogenesis and energy metabolism, and activating PPAR-RXR and pexophagy to promote fatty acid β-oxidation. Furthermore, we uncover that High BCAA strongly activates ubiquitin-proteasome autophagy via downregulating UFMylation to trigger MAPK9-mediated DNL, fatty acid elongation and lipid droplet formation-related proteins ubiquitination degradation, activating PPAR-RXR and pexophagy mediated fatty acid β-oxidation and lipolysis. Together, our data highlight moderating intake of high BCAA by inhibiting the AHR/MAPK9 are promising new strategies in NAFLD and FLHS treatment.

Unveiling the innovative green synthesis mechanism of selenium nanoparticles by exploiting intracellular protein elongation factor Tu from Bacillus paramycoides.

Selenium nanoparticles (SeNPs) have garnered extensive research interest and shown promising applications across diverse fields owing to their distinctive properties, including antioxidant, anticancer, and antibacterial activity (Ojeda et al., 2020; Qu et al., 2023; Zambonino et al., 2021, 2023). Among the various approaches employed for SeNP synthesis, green synthesis has emerged as a noteworthy and eco-friendly methodology. Keshtmand et al. (2023) underscored the significance of green-synthesized SeNPs, presenting a compelling avenue in this domain. This innovative strategy harnesses the potential of natural resources, such as plant extracts or microorganisms, to facilitate the production of SeNPs.

Genome-Wide Analysis of the SRPP/REF Gene Family in Taraxacum kok-saghyz Provides Insights into Its Expression Patterns in Response to Ethylene and Methyl Jasmonate Treatments.

Taraxacum kok-saghyz (TKS) is a model plant and a potential rubber-producing crop for the study of natural rubber (NR) biosynthesis. The precise analysis of the NR biosynthesis mechanism is an important theoretical basis for improving rubber yield. The small rubber particle protein (SRPP) and rubber elongation factor (REF) are located in the membrane of rubber particles and play crucial roles in rubber biosynthesis. However, the specific functions of the SRPP/REF gene family in the rubber biosynthesis mechanism have not been fully resolved. In this study, we performed a genome-wide identification of the 10 TkSRPP and 2 TkREF genes' family members of Russian dandelion and a comprehensive investigation on the evolution of the ethylene/methyl jasmonate-induced expression of the SRPP/REF gene family in TKS. Based on phylogenetic analysis, 12 TkSRPP/REFs proteins were divided into five subclades. Our study revealed one functional domain and 10 motifs in these proteins. The SRPP/REF protein sequences all contain typical REF structural domains and belong to the same superfamily. Members of this family are most closely related to the orthologous species T. mongolicum and share the same distribution pattern of SRPP/REF genes in T. mongolicum and L. sativa, both of which belong to the family Asteraceae. Collinearity analysis showed that segmental duplication events played a key role in the expansion of the TkSRPP/REFs gene family. The expression levels of most TkSRPP/REF members were significantly increased in different tissues of T. kok-saghyz after induction with ethylene and methyl jasmonate. These results will provide a theoretical basis for the selection of candidate genes for the molecular breeding of T. kok-saghyz and the precise resolution of the mechanism of natural rubber production.

Formulation and Quality Study of Mocaf Substitute Noodles with the Addition of Multigrain

This study aimed to determine the optimum formula and the characteristics of mocaf and multigrain (sorghum, kidney beans, mung beans) based noodle products. A design expert determined the optimum formula for the noodles. The noodles were tested for their gelatinization profile and tensile elongation profiles, protein content, and sensory evaluation using descriptive and preference tests to determine the optimum formula. The optimum formula was then compared to the noodle with the highest protein content for its nutrient, color, cooking properties, energy, fiber, and mineral content. The different formulas of multigrain noodles significantly affected the gelatinization and tensile elongation profiles. The higher content of mung beans and kidney beans increased the protein content. Multigrain noodles were accepted by consumers. The optimum formula for the multigrain noodle was 0% sorghum, 6.14% kidney beans, and 8.86% mung beans. Noodles with the highest protein content contained carbohydrates (83-84%), protein (7-8%), moisture content (6-8%), fat (0.3-0.5%), and ash (1-1.2%). The total energy of the noodles was about 365-372 Kcal/100g, while the energy from fat was about 2.75-4.46 Kcal/100g. These noodles contained high dietary fiber, consisting of 1.33-4.09% of soluble dietary fiber and 7.1-9.78% of insoluble dietary fiber. They had color and cooking properties comparable to other noodles that existed. The major minerals found in the noodles were potassium and sodium, followed by magnesium, calcium, iron, and zinc, respectively.

Reversing the relative time courses of the peptide bond reaction with oligopeptides of different lengths and charged amino acid distributions in the ribosome exit tunnel

The kinetics of the protein elongation cycle by the ribosome depends on intertwined factors. One of these factors is the electrostatic interaction of the nascent protein with the ribosome exit tunnel. In this computational biology theoretical study, we focus on the rate of the peptide bond formation and its dependence on the ribosome exit tunnel electrostatic potential profile. We quantitatively predict how oligopeptides of variable lengths can affect the peptide bond formation rate. We applied the Michaelis-Menten model as previously extended to incorporate the mechano-biochemical effects of forces on the rate of reaction at the catalytic site of the ribosome. For a given pair of carboxy-terminal amino acid substrate at the P- and an aminoacyl-tRNA at the A-sites, the relative time courses of the peptide bond formation reaction can be reversed depending on the oligopeptide sequence embedded in the tunnel and their variable lengths from the P-site. The reversal is predicted to occur from a shift in positions of charged amino acids upstream in the oligopeptidyl-tRNA at the P-site. The position shift must be adjusted by clever design of the oligopeptide probes using the electrostatic potential profile along the exit tunnel axial path. These predicted quantitative results bring strong evidence of the importance and relative contribution of the electrostatic interaction of the ribosome exit tunnel with the nascent peptide chain during elongation.

Evolutionary tinkering enriches the hierarchical and nested structures in amino acid sequences

Genetic information often exhibits hierarchical and nested relationships, achieved through the reuse of repetitive subsequences such as duplicons and transposable elements, a concept termed “evolutionary tinkering” by François Jacob. Current bioinformatics tools often struggle to capture these, particularly the nested, relationships. To address this, we utilized ladderpath, an approach within the broader category of algorithmic information theory, introducing two key measures: order rate η for characterizing sequence pattern repetitions and regularities, and ladderpath-complexity κ for assessing hierarchical and nested richness. Our analysis of amino acid sequences revealed that humans have more sequences with higher κ values, and proteins with many intrinsically disordered regions exhibit increased η values. Additionally, it was found that extremely long sequences with low η are rare. We hypothesize that this arises from varied duplication and mutation frequencies across different evolutionary stages, which in turn suggests a zigzag pattern for the evolution of protein complexity. This is supported by simulations and studies of protein families such as ubiquitin and NBPF, implying species-specific or environment-influenced protein elongation strategies. The ladderpath approach offers a quantitative lens to understand evolutionary tinkering and reuse, shedding light on the generative aspects of biological structures. Published by the American Physical Society 2024

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Evolutionary Conservation in Protein-Protein Interactions and Structures of the Elongator Sub-Complex ELP456 from Arabidopsis and Yeast.

The Elongator complex plays a pivotal role in the wobble uridine modification of the tRNA anticodon. Comprising two sets of six distinct subunits, namely, Elongator proteins (ELP1-ELP6) and associated proteins, the holo-Elongator complex demonstrates remarkable functional and structural conservation across eukaryotes. However, the precise details of the evolutionary conservation of the holo-Elongator complex and its individual sub-complexes (i.e., ELP123; ELP456) in plants remain limited. In this study, we conducted an in vivo analysis of protein-protein interactions among Arabidopsis ELP4, ELP5, and ELP6 proteins. Additionally, we predicted their structural configurations and performed a comparative analysis with the structure of the yeast Elp456 sub-complex. Protein-protein interaction analysis revealed that AtELP4 interacts with AtELP6 but not directly with AtELP5. Furthermore, we found that the Arabidopsis Elongator-associated protein, Deformed Roots and Leaves 1 (DRL1), did not directly bind to AtELP proteins. The structural comparison of the ELP456 sub-complex between Arabidopsis and yeast demonstrated high similarity, encompassing the RecA-ATPase fold and the positions of hydrogen bonds, despite their relatively low sequence homology. Our findings suggest that Arabidopsis ELP4, ELP5, and ELP6 proteins form a heterotrimer, with ELP6 serving as a bridge, indicating high structural conservation between the ELP456 sub-complexes from Arabidopsis and yeast.

Identification and Validation of Novel Metastasis-Related Immune Gene Signature in Breast Cancer.

Distant metastasis remains the leading cause of death among patients with breast cancer (BRCA). The process of cancer metastasis involves multiple mechanisms, including compromised immune system. However, not all genes involved in immune function have been comprehensively identified. Firstly 1623 BRCA samples, including transcriptome sequencing and clinical information, were acquired from Gene Expression Omnibus (GSE102818, GSE45255, GSE86166) and The Cancer Genome Atlas-BRCA (TCGA-BRCA) dataset. Subsequently, weighted gene co-expression network analysis (WGCNA) was performed using the GSE102818 dataset to identify the most relevant module to the metastasis of BRCA. Besides, ConsensusClusterPlus was applied to divide TCGA-BRCA patients into two subgroups (G1 and G2). In the meantime, the least absolute shrinkage and selection operator (LASSO) regression analysis was used to construct a metastasis-related immune genes (MRIGs)_score to predict the metastasis and progression of cancer. Importantly, the expression of vital genes was validated through reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The expression pattern of 76 MRIGs screened by WGCNA divided TCGA-BRCA patients into two subgroups (G1 and G2), and the prognosis of G1 group was worse. Also, G1 exhibited a higher mRNA expression level based on stemness index score and Tumor Immune Dysfunction and Exclusion score. In addition, higher MRIGs_score represented the higher probability of progression in BRCA patients. It was worth mentioning that the patients in the G1 group had a high MRIGs_score than those in the G2 group. Importantly, the results of RT-qPCR and IHC demonstrated that fasciculation and elongation protein zeta 1 (FEZ1) and insulin-like growth factor 2 receptor (IGF2R) were risk factors, while interleukin (IL)-1 receptor antagonist (IL1RN) was a protective factor. Our study revealed a prognostic model composed of eight immune related genes that could predict the metastasis and progression of BRCA. Higher score represented higher metastasis probability. Besides, the consistency of key genes in BRCA tissue and bioinformatics analysis results from mRNA and protein levels was verified.

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background.

Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.IMPORTANCEThis study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein elongation factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Unraveling the crystal structure of the HpaA adhesin: insights into cell adhesion function and epitope localization of a Helicobacter pylori vaccine candidate.

Helicobacter pylori is a bacterium that can cause chronic gastritis, peptic ulcers, and gastric cancers. The bacterium adheres to the lining of the stomach using proteins called adhesins. One of these proteins, HpaA, is particularly important for H. pylori colonization and is considered a promising vaccine candidate against H. pylori infections. In this work, we determined the atomic structure of HpaA, identifying a characteristic protein fold to the Helicobacter family and delineating specific amino acids that are crucial to support the attachment to the gastric cells. Additionally, we discovered that HpaA can trigger the production of TNF-α, a proinflammatory molecule, in macrophages. These findings provide valuable insights into how H. pylori causes disease and suggest that HpaA has a dual role in both attachment and immune activation. This knowledge could contribute to the development of improved vaccine strategies for preventing H. pylori infections.

Proteomic analysis revealed the function of PoElp3 in development, pathogenicity, and autophagy through the tRNA-mediated translation efficiency in the rice blast fungus1

The Elongator complex is conserved in a wide range of species and plays crucial roles in diverse cellular processes. We have previously shown that the Elongator protein PoELp3 was involved in the asexual development, pathogenicity, and autophagy of the rice blast fungus. In this study, we further revealed that PoElp3 functions via tRNA-mediated protein integrity. Phenotypic analyses revealed that overexpression of two of the tRNAs, tK(UUU) and tQ(UUG) could rescue the defects in ΔPoelp3 strain. TMT-based proteomic and transcriptional analyses demonstrated that 386 proteins were down-regulated in ΔPoelp3 strain compared with wild type strain Guy11, in a transcription-independent manner. Codon usage assays revealed an enrichment of Glutamine CAA-biased mRNA in the 386 proteins compared with the 70-15 genome. In addition to those reported previously, we also found that PoErp9, a sphingolipid C9-methyltransferase, was down-regulated in the ΔPoelp3 strain. Through an ILV2-specific integration of PoERP9-GFP into the wild type and ΔPoelp3 strain, we were able to show that PoErp9 was positively regulated by PoElp3 translationally but not transcriptionally. Functional analyses revealed that PoErp9 was involved in the fungal growth, conidial development, pathogenicity, and TOR-related autophagy homeostasis in P. oryzae. Taken together, our results suggested that PoElp3 acts through the tRNA-mediated translational efficiency to regulate asexual development, pathogenicity, sphingolipid metabolism, and autophagy in the rice blast fungus.

An integrative framework for clinical diagnosis and knowledge discovery from exome sequencing data

Non-silent single nucleotide genetic variants, like nonsense changes and insertion-deletion variants, that affect protein function and length substantially are prevalent and are frequently misclassified. The low sensitivity and specificity of existing variant effect predictors for nonsense and indel variations restrict their use in clinical applications. We propose the Pathogenic Mutation Prediction (PMPred) method to predict the pathogenicity of single nucleotide variations, which impair protein function by prematurely terminating a protein's elongation during its synthesis. The prediction starts by monitoring functional effects (Gene Ontology annotation changes) of the change in sequence, using an existing ensemble machine learning model (UniGOPred). This, in turn, reveals the mutations that significantly deviate functionally from the wild-type sequence. We have identified novel harmful mutations in patient data and present them as motivating case studies. We also show that our method has increased sensitivity and specificity compared to state-of-the-art, especially in single nucleotide variations that produce large functional changes in the final protein. As further validation, we have done a comparative docking study on such a variation that is misclassified by existing methods and, using the altered binding affinities, show how PMPred can correctly predict the pathogenicity when other tools miss it. PMPred is freely accessible as a web service at https://pmpred.kansil.org/, and the related code is available at https://github.com/kansil/PMPred.

Newly Recognized Genetic Tumor Syndromes of the CNS in the 5th WHO Classification: Imaging Overview with Genetic Updates.

The nervous system is commonly involved in a wide range of genetic tumor-predisposition syndromes. The classification of genetic tumor syndromes has evolved during the past years; however, it has now become clear that these syndromes can be categorized into a relatively small number of major mechanisms, which form the basis of the new 5th edition of the World Health Organization book (beta online version) on genetic tumor syndromes. For the first time, the World Health Organization has also included a separate chapter on genetic tumor syndromes in the latest edition of all the multisystem tumor series, including the 5th edition of CNS tumors. Our understanding of these syndromes has evolved rapidly since the previous edition (4th edition, 2016) with recognition of 8 new syndromes, including the following: Elongator protein complex-medulloblastoma syndrome, BRCA1-associated protein 1 tumor-predisposition syndrome, DICER1 syndrome, familial paraganglioma syndrome, melanoma-astrocytoma syndrome, Carney complex, Fanconi anemia, and familial retinoblastoma. This review provides a description of these new CNS tumor syndromes with a focus on imaging and genetic characteristics.

A novel stop-loss mutation in NKX2-2 gene as a cause of neonatal diabetes mellitus: molecular characterization and structural analysis.

To identifythe genetic etiology of neonatal diabetes in an infant and to elucidate the molecular mechanism of the identified mutation underlying the pathogenesis. Genetic analysis was carried out by sequencing of known etiological genes associated with NDM. Molecular characterization was performed by constructing aidentified mutation inNKX2-2 geneand functional aspects wastested using transactivation, protein expression, DNA binding, nuclear localization assays. Structural analysis was performed bymodeling the NKX2-2 protein structure. A novel homozygous frameshift mutationc.772delC, p.Q258SFs*59in the NKX2-2 gene was identified in a patient with neonatal diabetes. Functional studies revealed that this mutation resulted in an elongated protein sequence, affecting DNA binding activity and transcriptional function. Structural analysis suggested alterations in the protein's tertiary structure, likely contributing to its dysfunction. This study presents the first report of a stop-loss mutation in the NKX2-2 gene associated with NDM. Our findings emphasize the importance of functional and structural characterization to understand the biological consequences of such mutations. This comprehensive analysis provides insights into the molecular mechanisms underlying NDM and its clinical phenotype, which may aid in better diagnosis and management of patients with similar variants in the future.

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.

Stem cells from human exfoliated deciduous teeth rejuvenate the liver in naturally aged mice by improving ribosomal and mitochondrial proteins

Background aimsAging is accompanied by a decline in cellular proteome homeostasis, mitochondrial, and metabolic function. Mesenchymal stromal cell (MSC) therapies have been reported to extend lifespan and delay some age-related pathologies, yet the anti-aging rate and mechanisms remain unclear. Here, we investigated the effects and mechanism by transplantation of stem cells from human exfoliated deciduous teeth (SHED) into the naturally aged mice model. MethodsSHED were cultured in vitro and injected into mice by caudal vein. The in vivo imaging uncovered that SHED labeled by DiR dye mainly migrated to the liver, spleen, and lung organs of wild-type mice. As the main metabolic organ and SHED homing place, the liver was selected for proteomics and aging clock algorithm (LiverClock) analysis, which was constructed to estimate the proteomic pattern related to liver age state. ResultsAfter 6 months of continuous SHED injections, the liver proteomic pattern was reversed from senescent (∼30 months) to a youthful state (∼3 months), accompanied with upregulation of hepatocytes marker genes, anti-aging protein Klotho, a global improvement of liver functional pathways proteins, and a dramatic regulation of ribosomal and mitochondrial proteins, including upregulation of translation elongation and ribosome-sparing proteins Rpsa and Rplp0; elongation factors Eif4a1, Eef1b2, Eif5a; protein-folding chaperones Hsp90aa and Hspe1; ATP synthesis proteins Atp5b, Atp5o, Atp5j; and downregulation of most ribosomal proteins, suggesting that the proteome homeostasis destruction and mitochondria dysfunction in the aged mice liver might be relieved after SHED treatment. ConclusionsSHED treatment could dramatically relieve the senescent state of the aged liver, affect ribosome component proteins and upregulate the ribosomal biogenesis proteins in the aged mice liver. These results may help understand the improvements and mechanisms of SHED treatment in anti-aging.