Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity / 药物分析学报

Ribosomes are abundant,large RNA-protein complexes that are the sites of all protein synthesis in cells.Defects in ribosomal proteins(RPs),including proteoforms arising from genetic variations,alternative splicing of RNA transcripts,post-translational modifications and alterations of protein expression level,have been linked to a diverse range of diseases,including cancer and aging.Comprehensive character-ization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets.In the present work,using E.coli 70S RPs as an example,we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry(MS)system,and then applied it to the HeLa 80S ribosome.The results were complemented by a bottom-up approach.In total,50 out of 55 RPs were identified using the top-down approach.Among these,more than 30 RPs were found to have their N-terminal methionine removed.Additional modifications such as methylation,acetylation,and hydroxylation were also observed,and the modification sites were identified by bottom-up MS.In a HeLa 80S ribosomal sample,we identified 98 ribosomal proteoforms,among which multiple truncated 80S ribosomal proteoforms were observed,the type of information which is often overlooked by bottom-up experiments.Although their relevance to diseases is not yet known,the integration of top-down and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Anti-ribosomal P (anti-P) antibodies in patients with autoimmune hepatitis

ABSTRACT Objective Published studies have shown associations between anti-ribosomal P (anti-P) antibody and systemic lupus erythematosus with hepatic manifestations. This has been reported also in autoimmune hepatitis. However, the consistency of the latter association remains controversial. This study aimed to evaluate the frequency of anti-P antibodies in autoimmune hepatitis using two different immunoassays. Methods One-hundred and seventy-seven patients with autoimmune hepatitis were screened, and 142 were analyzed for anti-P antibody positivity. The samples were first analyzed using two different immunoassays: enzyme-linked immunosorbent assay (ELISA) and chemiluminescence and then compared with a group of 60 patients with systemic lupus erythematous. The positive samples were subjected to western blot analysis. Results Anti-P was found in 5/142 autoimmune hepatitis cases (3.5%) by chemiluminescence and in none by ELISA. Among the five chemiluminescence-positive autoimmune hepatitis samples, on anti-P western blot analysis one was negative, two were weakly positive, and two were positive. In contrast, anti-P was detected in 10/60 patients with systemic lupus erythematosus (16.7%) and presented higher chemiluminescence units than the autoimmune hepatitis samples. Conclusion A low frequency of anti-P antibodies was observed in autoimmune hepatitis, suggesting that this test is not useful for the diagnosis or management of this disease.

Ribosomal proteins and tumors / 国际肿瘤学杂志

The ribosome composed of ribosomal RNA and ribosomal proteins is well documented to be an important organelle for protein synthesis.Ribosomal proteins play crucial roles in protein translation.The dysregulation of ribosomal proteins and ribosomal RNA expression activates ribosomal proteins' extraribosomal functions which is believed to play important roles in the tumorigenesis.It is possible that ribosomal proteins may serve as biomarkers or promising targets for the early diagonosis and therapy of tumor diseases.

Ribosomal proteins and tumors / 国际肿瘤学杂志

The ribosome composed of ribosomal RNA and ribosomal proteins is well documented to be an important organelle for protein synthesis.Ribosomal proteins play crucial roles in protein translation.The dysregulation of ribosomal proteins and ribosomal RNA expression activates ribosomal proteins' extraribosomal functions which is believed to play important roles in the tumorigenesis.It is possible that ribosomal proteins may serve as biomarkers or promising targets for the early diagonosis and therapy of tumor diseases.

The difference expression and diagnostic value of RPL5 in papillary thyroid carcinoma of children and adults / 中华耳鼻咽喉头颈外科杂志

Objective@#To study the difference expression and diagnostic value of ribosomal protein L5 (RPL5) in papillary thyroid carcinoma (PTC) of children and adults.@*Methods@#Realtime-PCR was performed to detect the expression of RPL5 in 22 PTC tissues and 13 pericarcinous tissues. Receiver operating characteristic (ROC) curve and Youden's index were used to evaluate the diagnostic value of RPL5 in PTC of children and adults.@*Results@#The expression of RPL5 in PTC tissues was higher than in pericarcinous tissues. The area under curve (AUC) was 0.820 (P=0.001), and Youden′s index was 0.568. The expression of RPL5 in PTC of adults was higher than children (P<0.05). The AUC and Youden's index were respectively 0.721 (P=0.069) and 0.414 in children, whereas being respectively 0.896 (P=0.0005) and 0.709 in adults. RPL5 in diagnosis of PTC of adults was better than CK19, Galectin-3 and TPO, which are commonly used for the pathologic diagnosis of PTC.@*Conclusion@#The expression of RPL5 in PTC is higher than pericarcinous tissues, and its expression in PTC of adults is higher than children. Furthermore, PTC is a potential indicator for diagnosis of PTC.

The expression of RPS15a in colorectal cancer tissues and the effects of silencing RPS15a on the proliferation and apoptosis of colon cancer RKO cells / 肿瘤

Objective: To investigate the role of ribosomal protein S15a (RPS15a)in colorectal cancer.Methods: The expressions of RPS15a in 120 specimens of colorectalcancer tissues and the para-cancerous tissues as well as 120specimens of colorectal adenoma tissues were detected byimmunohistochemistry. The proliferation, cell cycle and apoptosisof colon cancer RKO cells after infection with recombinant lentivirusRPS15a-siRNA were detected by Cellomics cell counting assay, colony formation assay and FCM, respectively.Results: The positive rate of RPS15a expression in colorectal cancer tissues was significantlyhigher than those in the colorectal adenoma tissues (86.7% vs 25.8%, P < 0.001) andthe para-cancerous tissues (86.7% vs 11.7%, P < 0.001). The expression of RPS15a wassignificantly correlated with the TNM stage (P = 0.033) and the tumor differentiation (P <0.001) of colorectal cancer. RPS 15a silencing significantly suppressed the proliferation (P <0.01) and colony formation (P < 0.01) of the RKO cells, induced apoptosis (P < 0.01), andarrested the cell cycle at G2/M phase (P < 0.01).Conclusion: The expression of RPS15a in colorectal cancer tissues is higher than those in thecolorectal adenoma tissues and the para-cancerous tissues. Down-regulation of RPS 15aexpression can repress the proliferation and induce the apoptosis of RKO cells.

A cfr-positive clinical staphylococcal isolate from India with multiple mechanisms of linezolid-resistance.

Background & objectives: Linezolid, a member of the oxazolidinone class of antibiotics, has been an effective therapeutic option to treat severe infections caused by multidrug resistant Gram positive bacteria. Emergence of linezolid resistant clinical strains is a serious issue in the healthcare settings worldwide. We report here the molecular characterization of a linezolid resistant clinical isolate of Staphylococcus haemolyticus from India. Methods: The species of the clinical isolate was identified by 16S rRNA gene sequencing. The minimum inhibitory concentrations (MICs) of linezolid, clindamycin, chloramphenicol and oxacillin were determined by E-test method. To elucidate the mechanism of linezolid-resistance, presence of cfr gene (chloramphenicol florfenicol resistance) and mutations in 23S rRNA and ribosomal proteins (L3, L4 and L22) were investigated. Staphylococcal Cassette Chromosome mec (SCCmec) typing was performed by multiplex PCR. Results: The study documented a rare clinical S. haemolyticus strain with three independent mechanisms of linezolid-resistance. The strain carried cfr gene, the only known transmissible mechanism of linezolid-resistance. The strain also possessed resistance-conferring mutations such as G2576T in domain V of 23S rRNA gene and Met156Thr in L3 ribosomal protein. The other ribosomal proteins (L4 and L22) did not exhibit mutations accountable for linezolid-resistance. Restriction digestion by NheI revealed that all the alleles of 23S rRNA gene were mutated. The isolate showed elevated MIC values (>256 μg ml-1) of linezolid, clindamycin, chloramphenicol and oxacillin. Methicillin resistance was conferred by type I SCCmec element. The strain also harboured lsa(B) gene which encodes an ABC transporter that can efflux clindamycin. Interpretation & conclusions: The present study reports the first clinical strain from India with transmissible and multiple mechanisms of linezolid-resistance. Judicious use of linezolid in clinical practice and proper surveillance of cfr-positive strains are of utmost importance to safeguard the efficacy of linezolid.

Avaliação da capacidade protetora de antígenos recombinantes contra a Leishmaniose Tegumentar / Protective capacity of recombinant antigens against tegumentary leishmaniasis

A leishmaniose é uma doença de escala global, que afeta 12 milhões de pessoas e pode causar um espectro de doenças que vai desde a forma cutânea localizada, que tende para a cura espontânea, até a forma visceral que é fatal. Apesar da gravidade da doença, até o momento não existe uma vacina efetiva para prevenir a leishmaniose. Dentre os antígenos promissores para o desenvolvimento de uma vacina, destacam-se as proteínas ribossomais (S4, S6, L3 e L5) e a KMP-11, uma proteína de superfície presente nos membros da família tripanosomatidae. Nosso estudo consistiu em avaliar os efeitos da imunização com estes antígenos frente ao desafio com L. major e com L. braziliensis, empregando modelos experimentais de infecção. Primeiramente, avaliamos a capacidade protetora dos antígenos ribossomais frente à infecção por L. major. Dos quatro antígenos avaliados, apenas L3 ou L5 foram capazes de prevenir o desenvolvimento da lesão e de diminuir a carga parasitária. A vacinação de camundongos com estes antígenos, na presença de CpG, induziu um perfil de resposta Th1, com elevada produção de IFN-γ, baixa produção de IL-10 e presença de anticorpos IgG2a. Em seguida, avaliamos a capacidade protetora dos antígenos L3 e L5...

Leishmaniasis is a global disease affecting 12 million people and can cause diseases that range from self-healing localized cutaneous leishmaniasis to fatal visceral leishmaniasis. Despite the severity of the disease, there is no effective vaccine to prevent leishmaniasis. Among the promising antigens for the development of a vaccine, stand out the ribosomal proteins (S4, S6, L3, and L5) and KMP-11, a surface protein, widely found in the members of family Trypanosomatidae. Our study evaluated the effects of immunization with these antigens upon challenge with L. major and L. braziliensis, employing the experimental models of infection. First, we evaluated the protective ability of ribosomal antigens to infection by L. major. Among the four antigens examined only L3 or L5...

Identificación de una nueva proteína en Leishmania (Viannia) peruviana

El análisis de la secuencia nucleotídica y aminoacídica de un clon de la biblioteca de expresión en fago λgt11 de Leishmania (Viannia) peruviana, estableció identidad parcial con los genes de las proteínas acídicas ribosomales P2 de Leishmania (Leishmania) infantum. Este hallazgo unido a ciertos dominios geonómicos conservados, sugeridos de la comparación de 14 secuencias de otras proteínas P1 eucarióticas, confirman que la secuencia del inserto de clon codifica la proteína acídica ribosomal P1 de L. (V.) peruviana denominada LpP1. Este es el primer reporte sobre este tipo de proteína en el género Leishmania.

Nucleotidic and aminoacidic sequence analysis from a clone of a phage λgt11 obtained from a expression vector library of Leishmania (Viannia) peruviana, established partial identity with the genes of the acidic ribosomal proteins P2 of Leishmania (Leishmania) infantum. These molecular findings along with certain conserved genomic domains, suggested by comparison of 14 sequences of eukaryotic P1 proteins, confirmed that the insert of the clone codes for acidic ribosomal protein P1 of L (V) peruviana refered as LpP1. This is the first report about this type of protein in Leishmania genus.

Molecular interactions between ribosomal proteins—An analysis of S7–S9, S7–S19, S9–S19 and S7–S9–S19 interactions.

Ribosomal proteins S7, S9 and S 19 from Escherichia coli have been studied by the sedimentation equilibrium technique for possible intermolecular interaction between pairs of proteins as well as in a mixture of 3 proteins. The proteins were isolated to a purity greater than 95% and were characterized in the reconstitution buffer. It was observed that none of the proteins has a tendency to self-associate in the concentration range studied in the temperature range 3–6°C. Protein S9 behaves differently in the presence of other proteins. Analysis of the sedimentation equilibrium data for S7–S9, S9– S19 and S7–S9–S19 complexes revealed the need for considering the presence of a component of higher molecular weight in the system along with the monomers and their complexes to provide a meaningful curve-fitting of the data. Proteins S7 and S19 were found to interact with an equilibrium constant of association of 3 ± 2 × 104 M–1 at 3°C with a Gibbs free energy of interaction ΔG° of -5·7 kcal/mol. These data are useful for the consideration of the stabilization of the 30S subunit through protein-protein interactions and also help in building a topographical model of the proteins of the small subunit from an energetics point of view.

Cloning, expression and purification of human MRPS17 cDNA in Escherichia coli / 解放军医学杂志

Objective To clone, express and purificate human MRPS17 cDNA in Escherichia coli (E. coli). Methods MRPS17 cDNA was obtained from total RNA isolated from primary cultured human hair papillary cells by RT-PCR and sequencing method, and then it was inserted into prokaryotic expression vector pET28a for the IPTG-induced expression in E. coli BL21 (DE3). The expression product fused with 6?His at C-terminal was analyzed by Western blotting, and purified by using Ni 2+-NTA ion exchange resin. The purity of MRPS17 protein was analyzed by SDS-PAGE. Results Human MRPS17 cDNA was obtained, and the expression plasmid pET28a-MRPS17 was constructed successfully. Western blot analysis showed that human MRPS17 with 13kD molecular weight was expressed in E. coli at 20℃. The purity of the recombinant MRPS17 was more than 90% after purification using Ni 2+-2NTA ion exchange resin. Conclusion The sequence of MRPS17 cDNA was consistent with the known sequence from the Genbank. MRPS17 is successfully induced and expressed in E. coli.