RESUMEN

Post-transcriptional modifications (PTMs) are pivotal in the regulation of gene expression, and pseudouridylation is emerging as a critical player. This modification, facilitated by enzymes such as NOB1 (PNO1), is integral to ribosome biogenesis. PNO1, in collaboration with the NIN1/RPN12 binding protein 1 homolog (NOB1), is vital for the maturation of ribosomes, transitioning 20S pre-rRNA into functional 18S rRNA. Recent studies have highlighted PNO1's potential involvement in cancer progression; however, its underlying mechanisms remain unclear. Relentless growth characterizing cancer underscores the burgeoning significance of epitranscriptomic modifications, including pseudouridylation, in oncogenesis. Given PNO1's emerging role, it is imperative to delineate its contribution to cancer development to identify novel therapeutic interventions. This review summarizes the current literature regarding the role of PNO1 in cancer progression and its molecular underpinnings in oncogenesis. Overexpression of PNO1 was associated with unfavorable prognosis and increased tumor malignancy. At the molecular level, PNO1 facilitates cancer progression by modulating mRNA stability, alternative splicing, and translation efficiency. Its role in pseudouridylation of oncogenic and tumor-suppressor transcripts further underscores its significance in cancer biology. Although disruption of ribosome biogenesis is known to precipitate oncogenesis, the precise mechanisms by which these alterations contribute to cancer remain unclear. This review elucidates the intricate process of ribosomal small subunit maturation, highlighting the roles of crucial ribosomal proteins (RPs) and RNA-binding proteins (RBPs) as well as the positioning and function of NOB1 and PNO1 within the 40S subunit. The involvement of these components in the maturation of the small subunit (SSU) and their significance in the context of cancer therapeutics has been thoroughly explored. PNO1's burgeoning significance in oncology makes it a potential target for cancer therapies. Strategies aimed at modulating PNO1-mediated pseudouridylation may provide new avenues for cancer treatment. However, further research is essential to unravel the complete spectrum of PNO1 mechanisms in cancer and harness this knowledge for the development of targeted and more efficacious anticancer therapies.

RESUMEN

Flaviviral RNA genomes are composed of discrete RNA structural units arranged in an ordered fashion and grouped into complex folded domains that regulate essential viral functions, e.g. replication and translation. This is achieved by adjusting the overall structure of the RNA genome via the establishment of inter- and intramolecular interactions. Translation regulation is likely the main process controlling flaviviral gene expression. Although the genomic 3' UTR is a key player in this regulation, little is known about the molecular mechanisms underlying this role. The present work provides evidence for the specific recruitment of the 40S ribosomal subunit by the 3' UTR of the West Nile virus RNA genome, showing that the joint action of both genomic ends contributes the positioning of the 40S subunit at the 5' end. The combination of structural mapping techniques revealed specific conformational requirements at the 3' UTR for 40S binding, involving the highly conserved SL-III, 5'DB, 3'DB and 3'SL elements, all involved in the translation regulation. These results point to the 40S subunit as a bridge to ensure cross-talk between both genomic ends during viral translation and support a link between 40S recruitment by the 3' UTR and translation control.

Asunto(s)

Flavivirus , Virus del Nilo Occidental , Virus del Nilo Occidental/genética , Regiones no Traducidas 3' , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Flavivirus/genética , Genómica , ARN Viral/metabolismo , Replicación Viral

RESUMEN

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.

Asunto(s)

Iniciación de la Cadena Peptídica Traduccional , Ribosomas , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Ribosomas/metabolismo , Sistemas de Lectura , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ADN Polimerasa gamma/genética , ADN Polimerasa gamma/metabolismo

RESUMEN

Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a limited number of organisms has been resolved to date; it especially concerns plant ribosomes and ribosomal subunits. Here, we report a high-resolution cryo-electron microscopy reconstruction of the small subunit of the Triticum aestivum (common wheat) cytoplasmic ribosome. A detailed atomic model was built that includes the majority of the rRNA and some of the protein modifications. The analysis of the obtained data revealed structural peculiarities of the 40S subunit in the monocot plant ribosome. We applied the 3D Flexible Refinement approach to analyze the internal mobility of the 40S subunit and succeeded in decomposing it into four major motions, describing rotations of the head domain and a shift in the massive rRNA expansion segment. It was shown that these motions are almost uncorrelated and that the 40S subunit is flexible enough to spontaneously adopt any conformation it takes as a part of a translating ribosome or ribosomal complex. Here, we introduce the first high-resolution structure of an isolated plant 40S subunit and the first quantitative analysis of the flexibility of small ribosomal subunits, hoping that it will help in studying various aspects of ribosome functioning.

Asunto(s)

Subunidades Ribosómicas Pequeñas de Eucariotas , Ribosomas , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Microscopía por Crioelectrón , Ribosomas/metabolismo , ARN Ribosómico/metabolismo , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo

RESUMEN

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.

Asunto(s)

Proteínas de Unión al ADN , Biosíntesis de Proteínas , Proteínas Ribosómicas , Subunidades Ribosómicas Pequeñas de Eucariotas , Factores de Transcripción , Nucléolo Celular/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Humanos , Animales , Ratas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on ß-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , ARN Mensajero/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas

RESUMEN

All betacoronaviruses (ß-CoVs) encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts host gene expression. Among the ß-CoV family, MERS-CoV is the most distantly related member to SARS-CoV-2, and the mechanism for host translation inhibition by MERS-CoV Nsp1 remains controversial. Herein, we show that MERS-CoV Nsp1 directly interacts with the 40S ribosomal subunit. Using cryogenic electron microscopy (cryo-EM), we report a 2.6-Å structure of the MERS-CoV Nsp1 bound to the human 40S ribosomal subunit. The extensive interactions between C-terminal domain of MERS-CoV Nsp1 and the mRNA entry channel of the 40S ribosomal subunit are critical for its translation inhibition function. This mechanism of MERS-CoV Nsp1 is strikingly similar to SARS-CoV and SARS-CoV-2 Nsp1, despite modest sequence conservation. Our results reveal that the mechanism of host translation inhibition is conserved across ß-CoVs and highlight a potential therapeutic target for the development of antivirals that broadly restrict ß-CoVs.

Asunto(s)

Coronavirus del Síndrome Respiratorio de Oriente Medio , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , SARS-CoV-2/genética , ARN Mensajero/metabolismo , Proteínas no Estructurales Virales/metabolismo

RESUMEN

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on ß-globin, EMCV IRES and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nucleotides downstream from the mRNA entrance indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of Nsp1-NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

RESUMEN

Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stress-induced small RNAs, which are 5'- and 3'-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5'-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 µM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5'- and 3'-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops.

RESUMEN

Diverse elements within the 5' untranslated region of an mRNA can influence the translation efficiency at the main AUG codon. We previously identified a core picornaviral like Y16X11-AUG motif with 16-nt polypyrimidine CU tract separated by an 11-nt spacer sequence from the 13th AUG codon, which is recognized as the preferred initiation site within the Triticum mosaic virus (TriMV) internal ribosome entry site (IRES) element. The motif is proposed to function as an internal ribosomal landing site at the designated start codon. Here, we exposed the cooperative role of multiple CU-rich segments flanking the TriMV YX-AUG motif to reach and drive internal initiation of translation at the preferred start site. We propose that these auxiliary domains may enhance the ribosome capacity and their delivery at proximity of the correct initiation site. These polypyrimidine tracts can be modulated with a cryptic AUG in a position-dependent manner to replace the native YX-AUG motif, and thus uncovering a new layer of control of start codon selection. In line with these observations, mass spectrometry analysis of proteins directly interacting with translationally impaired TriMV IRES mutants that bear these motifs indicated an enrichment in 40S and 60S ribosomal related proteins, revealing a new function of polypyrimidine tracts to regulate IRES-driven translation. Accessibility of these RNA regions for in trans interaction was validated by SHAPE analysis of the entire TriMV leader sequence and supported by the ability of anti-sense oligonucleotides designed to block the CU tracts accessibility to impair IRES activity. This is the first evidence that defines the core modular domains required for ribosomal recruitment and start codon selection in a complex, multi-AUG viral 5' UTR for translation in plants.

RESUMEN

Triple negative breast cancer (TNBC) lacks targeted treatment resulting in poor prognosis. Targeting overexpressing mesothelin (MSLN) using MSLN­specific T cells is an attractive treatment approach and the aim of the present study. The expression of MSLN in human TNBC paraffin sections was analyzed by immunohistochemistry. Lentiviral vector harbored granulocyte­macrophage colony stimulating factor (GM­CSF), interleukin­4 (IL­4) and MSLN cDNAs was constructed to generate self­differentiated myeloid­derived antigen­presenting­cells reactive against tumor expressing MSLN dendritic cell (MSLN­SmartDC) for MSLN­specific T cell activation. The results showed high MSLN in 32.8% of all breast cancer subtypes and 57% in TNBC. High MSLN was significantly associated with TNBC subtype and the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. MSLN­SmartDC exhibited comparable phenotype to DC generated by exogenous cytokine treatment and an addition of 40s ribosomal protein subunit 3 (RPS3), a toll­like receptor 4 ligand, enhanced DC maturation and function by upregulation of CD40, CD80 and CD83 expressions and IL­12p70 secretion. MSLN­specific CD8+CD69+ IFN­Î³+ T cells were detected in T cells activated by both MSLN­SmartDC and RPS3­MSLN­SmartDC. MSLN­specific T cells activated by these DCs showed more specific killing capability against naturally expressed MSLN­HCC70 and artificially MSLN­overexpressing MDA­MB­231 compared with parental MDA­MB­231 in both two dimensional (2D)­ and 3D­culture systems. In conclusion, the results demonstrated the efficacy of MSLN­SmartDC to promote MSLN­specific T cells response against TNBC and RPS3 can enhance the cytolytic activity of these T cells providing an alternative treatment approach for patients with TNBC.

Asunto(s)

Células Dendríticas , Mesotelina , Proteínas Ribosómicas , Linfocitos T , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Células Dendríticas/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Linfocitos T/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología

RESUMEN

The small ribosomal subunit protein Rps15/uS19 is involved in early nucleolar ribosome biogenesis and subsequent nuclear export of pre-40S particles to the cytoplasm. In addition, the C-terminal tail of Rps15 was suggested to play a role in mature ribosomes, namely during translation elongation. Here, we show that Rps15 not only functions in nucleolar ribosome assembly but also in cytoplasmic pre-40S maturation, which is indicated by a strong genetic interaction between Rps15 and the 40S assembly factor Ltv1. Specifically, mutations either in the globular or C-terminal domain of Rps15 when combined with the non-essential ltv1 null allele are lethal or display a strong growth defect. However, not only rps15 ltv1 double mutants but also single rps15 C-terminal deletion mutants exhibit an accumulation of the 20S pre-rRNA in the cytoplasm, indicative of a cytoplasmic pre-40S maturation defect. Since in pre-40S particles, the C-terminal tail of Rps15 is positioned between assembly factors Rio2 and Tsr1, we further tested whether Tsr1 is genetically linked to Rps15, which indeed could be demonstrated. Thus, the integrity of the Rps15 C-terminal tail plays an important role during late pre-40S maturation, perhaps in a quality control step to ensure that only 40S ribosomal subunits with functional Rps15 C-terminal tail can efficiently enter translation. As mutations in the C-terminal tail of human RPS15 have been observed in connection with chronic lymphocytic leukaemia, it is possible that apart from defects in translation, an impaired late pre-40S maturation step in the cytoplasm could also be a reason for this disease.

Asunto(s)

Proteínas Ribosómicas , Proteínas de Saccharomyces cerevisiae , Humanos , Biosíntesis de Proteínas , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Background: The Gail, Breast Cancer Surveillance Consortium (BCSC), and Tyrer-Cuzick breast cancer risk prediction models are recommended for use in primary care. Calculating breast cancer risk is particularly important for women in their 40s when deciding on mammography, with some guidelines recommending screening for those with 5-year risk similar to women age 50 (≥1.1%). Yet, little is known about risk estimate agreement among models for these women. Materials and Methods: Four hundred nine Boston-area women 40-49 years of age completed a risk questionnaire before a primary care visit to compute their breast cancer risk. The kappa statistic was used to examine when (1) Gail and BCSC agreed on 5-year risk ≥1.1%; (2) Gail estimated 5-year risk ≥1.7% and Tyrer-Cuzick estimated 10-year risk ≥5% (guideline thresholds for recommending prevention medications); and when (3) Gail and Tyrer-Cuzick agreed on lifetime risk ≥20% (threshold for breast MRI using Tyrer-Cuzick). Results: Participant mean age was 44.1 years, 56.7% were non-Hispanic white, and 7.8% had a first-degree relative with breast cancer. Of 266 with breast density information to estimate both Gail and BCSC, the models agreed on 5-year risk being ≥1.1% for 36 women, kappa = 0.34 (95% confidence interval: 0.23-0.45). Gail and Tyrer-Cuzick estimates led to agreement about prevention medications for 8 women, kappa 0.41 (0.20-0.61), and models agreed on lifetime risk ≥20% for 3 women, kappa 0.08 (-0.01 to 0.16). Conclusions: There is weak agreement on breast cancer risk estimates generated by risk models recommended for primary care. Using different models may lead to different clinical recommendations for women in their 40s.

Asunto(s)

Neoplasias de la Mama , Adulto , Mama , Densidad de la Mama , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Atención Primaria de Salud , Medición de Riesgo

RESUMEN

The two subunits of the eukaryotic ribosome are produced through quasi-independent pathways involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. One of the earliest intermediates of the small subunit (SSU or 40S) is the SSU processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors could induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S.

Asunto(s)

Metiltransferasas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Metiltransferasas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

Colostrum, the sole diet for newborns, is an emerging nutraceutical. To date, the chemopreventive effect of Bovine Colostrum against liver injury induced by the potent carcinogen, 7,12-dimethyl-Benz[a]anthracene (DMBA) is unexplored. Humans are daily exposed to DMBA which is a highly lipophilic environmental organic pollutant. The study aimed to investigate the hepatoprotective role of Bovine Colostrum against DMBA-induced hepatotoxicity using a rat model. Fifty male rats were divided into five groups; GI (control), GII (olive oil, vehicle for DMBA), GIII (DMBA), GIV (DMBA + Bovine Colostrum), GV (Bovine Colostrum). After 12 weeks, body weight changes and mortality were calculated. Histological and ultrastructural examinations of liver tissue were performed. Expressions of p53, TGFß2, TNF-α, S6K2, and c20orf20 were assessed by RT-PCR. Post-treatment with Bovine Colostrum increased both the body weight and the survival rate of rats treated with DMBA. In addition, remarkable protection against the pathological effect of DMBA was noted. Ultrastructurally, Bovine Colostrum ameliorated/prevented most of the toxic effects of DMBA on hepatocytes, including irregularities of nuclear envelope, clumping, and margination of heterochromatin aggregates, segregated nucleoli, and mitochondrial pleomorphism. Bovine Colostrum administration down-regulated p53, C20orf20, and S6K2 mRNA levels, and up-regulated TNF-α and TGFß2. In conclusion, Bovine Colostrum have a protective effect against DMBA-induced toxicity on the liver of albino rats. Consequently, Bovine Colostrum may prevent polycyclic aromatic hydrocarbons-induced hepatotoxicity and may be useful in promoting human health if supplemented in the diet.

RESUMEN

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

Asunto(s)

Acetiltransferasa D N-Terminal/metabolismo , Histonas/metabolismo , Humanos , Isoformas de Proteínas , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción

RESUMEN

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis. The ubiquitously expressed 40S ribosome protein SA (RPSA) is a multifunctional protein involved in the pathogenesis of multiple pathogens, especially those causing meningitis. However, the role of RPSA in SS2-induced meningitis is not clear. In this study, immunofluorescence staining revealed that SS2 infection promoted the intracellular transfer of RPSA to the surface of human cerebral microvascular endothelial cells (HCMECs). Moreover, SS2 infection promoted the accumulation of caveolin 1 (CAV1) and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface. SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by addition of methyl-ß-cyclodextrin (MßCD). Co-immunoprecipitation analysis demonstrated that α-enolase (ENO), a key virulence factor of SS2, interacted with RPSA, and promoted the interaction between RPSA and CAV1. Immunofluorescence staining, western blotting and flow cytometry analyses showed that damaged caveolae/rafts significantly enhanced ENO adhesion to HCMECs, promoted the "destruction" of RPSA by ENO, and enhanced the toxic effect of ENO on HCMECs. Importantly, these effects could be relieved upon the addition of cholesterol. We conclude that caveolae/rafts weaken the toxic effect of SS2 ENO on RPSA-mediated events in HCMECs. Our study has led to better understanding of the roles of RPSA and caveolae/rafts upon SS2 infection, and a new pathological role for RPSA in infection.

Asunto(s)

Caveolas/metabolismo , Caveolina 1/metabolismo , Células Endoteliales/microbiología , Fosfopiruvato Hidratasa/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Streptococcus suis/patogenicidad , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Fosfopiruvato Hidratasa/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Serogrupo , Streptococcus suis/clasificación , Streptococcus suis/enzimología , Factores de Virulencia

RESUMEN

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

Asunto(s)

Interacciones Huésped-Patógeno/genética , Lisina/genética , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , SARS-CoV-2/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Biología Computacional/métodos , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Lisina/metabolismo , Mutación , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , SARS-CoV-2/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas no Estructurales Virales/metabolismo

RESUMEN

Alcohol­associated hepatocellular carcinoma (HCC) is a subtype of HCC with poor prognosis. The present study aimed to identify key biomarkers for alcohol­associated HCC. The gene data profiles and corresponding clinical traits of patients with alcohol­associated HCC were downloaded from The Cancer Genome Atlas (TCGA) database. Firstly, good genes and good samples were identified, which were subsequently used to conduct weighted gene co­expression network analysis (WGCNA). Hub genes in the significant modules were selected following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and from constructing a protein­protein interaction (PPI) network. Real hub genes among hub genes were determined following progression, survival analysis and gene set enrichment analysis (GSEA), as well as reverse transcription­quantitative PCR and immunohistochemical staining of non­alcohol­ and alcohol­associated HCC samples. In total, 64 good samples of alcohol­associated HCC with height score <160 were selected, from which 15,195 good genes were identified and used to conduct WGCNA; 8 gene co­expressed modules were identified using WGCNA, while 3 modules (including pink, magenta and turquoise) were significantly associated with Child­Pugh score, T­stage and body weight. Following GO and KEGG analysis and construction of the PPI network, a total of 30 hub genes were identified in the aforementioned 3 gene co­expressed modules, while 16 hub genes (including AURKB, BUB1, BUB1B, CCNB1, CCNB2, CDC20, CDCA8, CDK1, PLK1, RPS5, RPS7, RPS8, RPS14, RPS27, RPSA and TOP2A) were associated with the development of alcohol­associated HCC, and had a significant prognosis value. Among these genes, only RPS8 was highly expressed in alcohol­associated HCC, but not in non­alcohol­associated HCC, while RPS5 was not significantly associated in either alcohol­ or non­alcohol­associated HCC. GSEA demonstrated that 10 pathways, including RNA polymerase and ribosome pathways were enriched in alcohol­associated HCC samples where RPS8 was highly expressed. Taken together, the results of the present study demonstrate that RPS8 may be a novel biomarker for the diagnosis of patients with alcohol­associated HCC.

Asunto(s)

Consumo de Bebidas Alcohólicas/efectos adversos , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Proteínas Ribosómicas/genética , Adulto , Anciano , Consumo de Bebidas Alcohólicas/patología , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Biología Computacional , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Hígado/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Pronóstico , Mapas de Interacción de Proteínas/genética , Análisis de Supervivencia

RESUMEN

In eukaryotes, the beak structure of 40S subunits is formed by the protrusion of the 18S rRNA helix 33 and three ribosomal proteins: eS10, eS12 and eS31. The exact role of these proteins in ribosome biogenesis is not well understood. While eS10 is an essential protein encoded by two paralogous genes in Saccharomyces cerevisiae, eS12 and eS31 are not essential proteins encoded by the single-copy genes RPS12 and UBI3, respectively. Here, we have analysed the contribution of yeast eS12 to ribosome biogenesis and compared it with that of eS31. Polysome analysis reveals that deletion of either RPS12 or UBI3 results in equivalent 40S deficits. Analysis of pre-rRNA processing indicates that eS12, akin to eS31, is required for efficient processing of 20S pre-rRNA to mature 18S rRNA. Moreover, we show that the 20S pre-rRNA accumulates within cytoplasmic pre-40S particles, as deduced from FISH experiments and the lack of nuclear retention of 40S subunit reporter proteins, in rps12∆ and ubi3∆ cells. However, these particles containing 20S pre-rRNA are not efficiently incorporated into polyribosomes. We also provide evidence for a genetic interaction between eS12 or eS31 and the late-acting 40S assembly factors Enp1 and Ltv1, which appears not to be linked to the dynamics of their association with or release from pre-40S particles in the absence of either eS12 or eS31. Finally, we show that eS12- and eS31-deficient ribosomes exhibit increased levels of translational misreading. Altogether, our data highlight distinct important roles of the beak region during ribosome assembly and function.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Fenotipo , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , Transporte de ARN , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo