Ribosome is an intracellular ribonucleoprotein particle that serves as the site of protein biosynthesis. Ribosomal dysfunction caused by mutations in genes encoding ribosomal proteins (RPs) and ribosome biogenesis factors (RBFs) can lead to a spectrum of diseases, collectively known as ribosomopathy. Phase separation is a thermodynamic process that produces multiple phases from a homogeneous mixture. The formation of membraneless organelles and intracellular structures, including ribosomes and nucleoli, cannot occur without the involvement of phase separation. Here, ribosome structure, biogenesis, and their relationship with ribosomopathy are systematically reviewed. The tissue specificity of ribosomopathy and the role of phase separation in ribosomopathy are particularly discussed, which may offer some clues for understanding the mechanisms of ribosomopathy. Then, some new ideas for the prevention, diagnosis, and treatment of ribosomopathy are provided.
Humans ; Ribosomes/physiology* ; Ribosomal Proteins/metabolism* ; Mutation ; Animals ; Cell Nucleolus/metabolism* ; Protein Biosynthesis ; Phase Separation

Loss of protein homeostasis is a hallmark of cellular senescence, and ribosome pausing plays a crucial role in the collapse of proteostasis. However, our understanding of ribosome pausing in senescent cells remains limited. In this study, we utilized ribosome profiling and G-quadruplex RNA immunoprecipitation sequencing techniques to explore the impact of RNA G-quadruplex (rG4) on the translation efficiency in senescent cells. Our results revealed a reduction in the translation efficiency of rG4-rich genes in senescent cells and demonstrated that rG4 structures within coding sequence can impede translation both in vivo and in vitro. Moreover, we observed a significant increase in the abundance of rG4 structures in senescent cells, and the stabilization of the rG4 structures further exacerbated cellular senescence. Mechanistically, the RNA helicase DHX9 functions as a key regulator of rG4 abundance, and its reduced expression in senescent cells contributing to increased ribosome pausing. Additionally, we also observed an increased abundance of rG4, an imbalance in protein homeostasis, and reduced DHX9 expression in aged mice. In summary, our findings reveal a novel biological role for rG4 and DHX9 in the regulation of translation and proteostasis, which may have implications for delaying cellular senescence and the aging process.
G-Quadruplexes ; Cellular Senescence ; Ribosomes/genetics* ; Humans ; Animals ; Mice ; DEAD-box RNA Helicases/genetics* ; Protein Biosynthesis ; RNA/chemistry* ; Neoplasm Proteins

Proteins are the basic building blocks of life. Studying the protein expression mechanism is essential for understanding the cellular organization principles and the development of biotechnology. Protein expression, involving transcription, translation, folding, and post-translational modification, is a complicatedly regulated process affected by various cellular components and sequence features of the expressed protein. Establishing protein expression models based on expression data is of great significance for probing into the regulatory factors and mechanisms of protein expression. Here we review the recent research progress in the mechanism models for quantitatively simulating the protein expression process and the prediction algorithms based on artificial intelligence for analyzing the regulatory factors. Chemical reaction network models have been developed to mathematically describe the elementary processes in protein expression and simulate the influences of various cellular components such as RNA polymerase and tRNA. However, the experimental determination of the huge number of model parameters is a big challenge. The main objective of data-driven AI models is to study the effects of protein/DNA sequences of the target protein on its expression, and subsequently optimize the sequences to improve protein expression. Methods combining mechanism models and AI models have the potential to deepen our understanding of protein expression processes, providing theoretical and technical support for the efficient production of high-value proteins and coordinate the regulation of different proteins.
Artificial Intelligence ; Proteins/metabolism* ; Algorithms ; Protein Biosynthesis

Endotoxins are common exogenous pyrogens. Excessive endotoxins in medical devices and injections can lead to serious consequences such as sepsis, septic shock, and even death. Therefore, endotoxin detection plays a crucial role in medical, pharmaceutical, and food sectors. The wide application of Limulus amebocyte lysate (LAL) has led to a sharp decline in the number of horseshoe crabs. Moreover, the LAL assay has limitations such as interbatch variations and difficulty in quantification. The recombinant factor C (rFC) assay is stable between batches, highly sensitive, and capable of quantitation, and thus it can be used as an alternative for the LAL assay. However, the high cost and complex procedures involved in producing recombinant factor C have limited the widespread application of this method. In order to simplify the preparation and reduce the production cost of recombinant factor C, this study focuses on the production of recombinant factor C based on the baculovirus expression system. Multiple measures such as a high-yield and anti-apoptotic vector qBac-IIIG, the optimal signal peptide, and the optimized codon were used to reach the goal of endotoxin detection with cell supernatant. This method simplifies the steps of protein purification. The sensitivity of the supernatant reached 0.05 EU/mL in a 1-L fermentation system, and 500 000 detecting reactions can be supported per liter of fermentation broth. This study increases the yield and activity of recombinant factor C, simplifies the procedures of protein purification, and reduces the cost, laying a foundation for the promotion and application of recombinant factor C in endotoxin detection.
Animals ; Recombinant Proteins/genetics* ; Horseshoe Crabs/chemistry* ; Baculoviridae/metabolism* ; Endotoxins/analysis* ; Protein C/biosynthesis* ; Genetic Vectors/genetics* ; Arthropod Proteins/genetics* ; Enzyme Precursors ; Serine Endopeptidases

As the most common type of protein glycosylation, N-glycosylation begins with the synthesis of the dolichol-linked oligosaccharide (DLO) precursor in the endoplasmic reticulum. The mannosyltransferase Alg1 catalyzes the addition of the first mannose molecule to DLO, serving as a key enzyme in this biochemical pathway. The defect of human ALG1 gene can lead to the congenital disorders of glycosylation (CDG), i.e., ALG1-CDG. Therefore, it is of great significance to establish the expression and activity assay system of Homo sapiens Alg1 (HsAlg1) in vitro. In this study, full-length plasmid pET28a-His6-HsAlg1 and transmembrane domain-lacking plasmid pET28a-His6-HsAlg1_23-464 were constructed and expressed in Escherichia coli, and the activity of recombinant HsAlg1 and HsAlg1_23-464 was measured by liquid chromatography tandem mass spectrometry (LC-MS) with dolichyl-pyrophosphate GlcNAc2 (DPGn2) as the substrate. The results showed that HsAlg1 had transglycosylation activity, while the activity decreased after protein purification, which was partially restored upon re-addition of membrane components. However, HsAlg1_23-464 was unable to catalyze glycosylation. The results indicate that the N-terminal transmembrane domain (TMD) of HsAlg1 plays an important role in the catalytic reaction. This study lays a foundation for further expression and activity analysis of ALG1-CDG-related mutants.
Humans ; Escherichia coli/metabolism* ; Mannosyltransferases/biosynthesis* ; Glycosylation ; Recombinant Proteins/metabolism* ; Protein Domains

The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

Phenylpyruvic acid (PPA) is used as a food and feed additive and has a wide range of applications in the pharmaceutical, chemical and other fields. At present, PPA is mainly produced by chemical synthesis. With the green transformation of the manufacturing industry, biotransformation will be a good alternative for PPA production. The L-amino acid deaminase (PmiLAAD) from Proteus mirabilis has been widely studied for the production of PPA. However, the low yield limits its industrial production. To further enhance the production of PPA and better meet industrial demands, a more efficient synthesis method for PPA was established. In this study, PmiLAAD was heterologously expressed in Escherichia coli. Subsequently, a colorimetric reaction method was established to screen the strains with high PPA production. The semi-rational design of PmiLAAD was carried out, and the obtained triple-site mutant V18 (V437I/S93C/E417A) showed a 35% increase in catalytic activity compared with the wild type. Meanwhile, the effect of N-terminal truncation on the catalytic activity of the V18 mutant was investigated. After the optimization of the whole-cell conditions for the obtained mutant V18-N7, fed-batch conversion was carried out in a 5-L fermenter, and 44.13 g/L of PPA was synthesized with a conversion rate of 88%, which showed certain potential for industrial application. This study lays foundation for the industrial production of phenylpyruvic acid and also offers insights into the biosynthesis of other chemicals.
Escherichia coli/metabolism* ; Proteus mirabilis/genetics* ; Phenylpyruvic Acids/metabolism* ; Protein Engineering/methods* ; Recombinant Proteins/biosynthesis* ; Bacterial Proteins/metabolism*

Protein tyrosine phosphatases (PTPs, EC 3.1.3.48) are key regulators of cellular processes, with the catalytic activity attributed to the conserved motif (H/V)CX₅R(S/T), where cysteine and arginine residues are critical. Previous studies revealed that alternative splicing of extracellular phosphatase mRNA precursors in Metarhizium anisopliae generated two distinct transcripts, with the longer sequence containing a novel HCPTPMLS motif resembling PTP signatures but lacking the arginine residue. To identify the novel signature motif and characterize its enzymatic properties, we heterologously expressed and purified both proteins in Pichia pastoris and comprehensively characterized their enzymatic properties. The protein containing the HCPTPMLS motif (designated as L-protein) exhibited the highest activity at pH 5.5 and a strong preference for pTyr substrates. Its phosphatase activity was inhibited by Ag⁺, Zn²⁺, Cu²⁺, molybdate, and tungstate, but enhanced by Ca²⁺ and EDTA. AcP101 (lacking HCPTPMLS) showed the maximal activity at pH 6.5 and a strong preference toward pNPP (P < 0.05), with the activity inhibited by NaF and tartrate, but enhanced by Mg²⁺ and Mn²⁺. Functional analysis confirmed that the L-protein retained the PTP activity despite the absence of arginine in its signature motif, while AcP101 functioned as an acid phosphatase. This study provides the first functional validation of an arginine-deficient PTP motif, expanding the definition of PTP signature motifs and offering new insights for phosphatase classification.
Metarhizium/genetics* ; Protein Tyrosine Phosphatases/chemistry* ; Amino Acid Motifs ; Recombinant Proteins/biosynthesis* ; Amino Acid Sequence ; Pichia/metabolism* ; Fungal Proteins/chemistry* ; Substrate Specificity ; Saccharomycetales

Penicillin G acylases (PGAs) are industrially important enzymes primarily used for the synthesis of first- and second-generation cephalosporins or penicillins. This study aims to establish a high-efficiency biosynthetic system for cefradine on the purpose of significantly enhancing its catalytic efficiency in cefradine synthesis and developing its potentials for industrial application. In this study, we identified and engineered penicillin G acylase and obtained a highly active mutant KsPGA M7(M168F/F313G) for the synthesis of cefradine. The mutant achieved a conversion rate over 95% in the scaled-up reaction. To validate its industrial applicability, we immobilized both the wild-type and mutant enzymes and applied them in continuous flow reactions, which achieved a space-time yield of 2 800 g/(L·d). This study lays a foundation for the future applications of penicillin G acylases in the industrial synthesis of cefradine.
Penicillin Amidase/biosynthesis* ; Protein Engineering/methods* ; Cephradine/metabolism* ; Escherichia coli/metabolism* ; Enzymes, Immobilized/metabolism* ; Recombinant Proteins/biosynthesis*

A primary hallmark of pathological cardiac hypertrophy is excess protein synthesis due to enhanced translational activity. However, regulatory mechanisms at the translational level under cardiac stress remain poorly understood. Here we examined the translational regulations in a mouse cardiac hypertrophy model induced by transaortic constriction (TAC) and explored the conservative networks versus the translatome pattern in human dilated cardiomyopathy (DCM). The results showed that the heart weight to body weight ratio was significantly elevated, and the ejection fraction and fractional shortening significantly decreased 8 weeks after TAC. Puromycin incorporation assay showed that TAC significantly increased protein synthesis rate in the left ventricle. RNA-seq revealed 1,632 differentially expressed genes showing functional enrichment in pathways including extracellular matrix remodeling, metabolic processes, and signaling cascades associated with pathological cardiomyocyte growth. When combined with ribosome profiling analysis, we revealed that translation efficiency (TE) of 1,495 genes was enhanced, while the TE of 933 genes was inhibited following TAC. In DCM patients, 1,354 genes were upregulated versus 1,213 genes were downregulated at the translation level. Although the majority of the genes were not shared between mouse and human, we identified 93 genes, including Nos3, Kcnj8, Adcy4, Itpr1, Fasn, Scd1, etc., with highly conserved translational regulations. These genes were remarkably associated with myocardial function, signal transduction, and energy metabolism, particularly related to cGMP-PKG signaling and fatty acid metabolism. Motif analysis revealed enriched regulatory elements in the 5' untranslated regions (5'UTRs) of transcripts with differential TE, which exhibited strong cross-species sequence conservation. Our study revealed novel regulatory mechanisms at the translational level in cardiac hypertrophy and identified conserved translation-sensitive targets with potential applications to treat cardiac hypertrophy and heart failure in the clinic.
Animals ; Humans ; Cardiomegaly/physiopathology* ; Ribosomes/physiology* ; Protein Biosynthesis/physiology* ; Mice ; Cardiomyopathy, Dilated/genetics* ; Ribosome Profiling