Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate incorporation of amino acids at the stop codon and production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. Herein, we report on the development a human cell model that can be used to determine whether rules can be developed using mass spectrometry that define the type of amino acid that is placed at a premature termination codon (PTC) during readthrough mediated by an aminoglycoside. The first PTC we analyzed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Despite of identifying a tryptic peptide with the incorporation of an R at codon 213 in the presence of the aminoglycoside, there were no other tryptic peptides detected across codon 213 that could be recovered; hence we constructed a more robust artificial PTC model. P53 expression plasmids were developed that incorporate a string of single synthetic TGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 121-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-TGA129 mutation, treated with Gentamicin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-TGA129; as expected based on the two-base pairing of the respective anticodons in the tRNA to UGA, with R being the most abundant. By contrast, incorporating the amber or ochre premature stop codons, TAA129 or TAG129 resulted in the incorporation of a Y or Q amino acid, again as expected based on the two base pairings to the anticodons, with Q being the most abundant. A reproducible non-canonical readthrough termination codon-skip event at the extreme C-terminus at codon 436 in the SBP-p53 fusion protein was detected which provided a novel assay for non-canonical readthrough at an extreme C-terminal PTC. The incorporation of amino acids at codons 127, 128, or 129 generally result in a p53 protein that is predicted to be 'unfolded' or inactive as defined by molecular dynamic simulations presumably because the production of mixed wild-type p53 and mutant oligomers are known to be inactive through dominant negative effects of the mutation. The data highlight the need to not only produce novel small molecules that can readthrough PTCs or C-terminal termination codons, but also the need to design methods to insert the required amino acid at the position that could result in a 'wild-type' functional protein.
- Publikační typ
- časopisecké články MeSH
The detection of HPV infection and microbial colonization in cervical lesions is currently done through PCR-based viral or bacterial DNA amplification. Our objective was to develop a methodology to expand the metaproteomic landscape of cervical disease and determine if protein biomarkers from both human and microbes could be detected in distinct cervical samples. This would lead to the development of multi-species proteomics, which includes protein-based lateral flow diagnostics that can define patterns of microbes and/or human proteins relevant to disease status. In this study, we collected both non-frozen tissue biopsy and exfoliative non-fixed cytology samples to assess the consistency of detecting human proteomic signatures between the cytology and biopsy samples. Our results show that proteomics using biopsies or cytologies can detect both human and microbial organisms. Across patients, Lumican and Galectin-1 were most highly expressed human proteins in the tissue biopsy, whilst IL-36 and IL-1RA were most highly expressed human proteins in the cytology. We also used mass spectrometry to assess microbial proteomes known to reside based on prior 16S rRNA gene signatures. Lactobacillus spp. was the most highly expressed proteome in patient samples and specific abundant Lactobacillus proteins were identified. These methodological approaches can be used in future metaproteomic clinical studies to interrogate the vaginal human and microbiome structure and metabolic diversity in cytologies or biopsies from the same patients who have pre-invasive cervical intraepithelial neoplasia, invasive cervical cancer, as well as in healthy controls to assess how human and pathogenic proteins may correlate with disease presence and severity.
- MeSH
- biologické markery * analýza metabolismus MeSH
- biopsie MeSH
- cervix uteri * mikrobiologie patologie MeSH
- dospělí MeSH
- galektin 1 metabolismus analýza genetika MeSH
- Lactobacillus MeSH
- lidé MeSH
- lumican MeSH
- mikrobiota MeSH
- nádory děložního čípku patologie mikrobiologie MeSH
- proteomika * metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.
- MeSH
- adenokarcinom * genetika metabolismus patologie MeSH
- lidé MeSH
- multiomika MeSH
- nádorové biomarkery * metabolismus genetika MeSH
- nádory jícnu * genetika metabolismus patologie MeSH
- posttranskripční úpravy RNA MeSH
- proteom metabolismus MeSH
- proteomika * metody MeSH
- regulace genové exprese u nádorů * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.
- MeSH
- antivirové látky farmakologie chemie metabolismus MeSH
- diferenciační antigeny * metabolismus chemie MeSH
- konformace proteinů * MeSH
- lidé MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus biosyntéza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.
Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.
BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.
Cardiovascular diseases, such as myocardial infarction, ischemic stroke, and pulmonary embolism, are the most common causes of disability and death worldwide. Blood clot hydrolysis by thrombolytic enzymes and thrombectomy are key clinical interventions. The most widely used thrombolytic enzyme is alteplase, which has been used in clinical practice since 1986. Another clinically used thrombolytic protein is tenecteplase, which has modified epitopes and engineered glycosylation sites, suggesting that carbohydrate modification in thrombolytic enzymes is a viable strategy for their improvement. This comprehensive review summarizes current knowledge on computational and experimental identification of glycosylation sites and glycan identity, together with methods used for their reengineering. Practical examples from previous studies focus on modification of glycosylations in thrombolytics, e.g., alteplase, tenecteplase, reteplase, urokinase, saruplase, and desmoteplase. Collected clinical data on these glycoproteins demonstrate the great potential of this engineering strategy. Outstanding combinatorics originating from multiple glycosylation sites and the vast variety of covalently attached glycan species can be addressed by directed evolution or rational design. Directed evolution pipelines would benefit from more efficient cell-free expression and high-throughput screening assays, while rational design must employ structure prediction by machine learning and in silico characterization by supercomputing. Perspectives on challenges and opportunities for improvement of thrombolytic enzymes by engineering and evolution of protein glycosylation are provided.
Calcium channel blockers are among the most commonly used agents in the treatment of cardiovascular diseases. There are several known side-effects associated with their long-term use, whereas other potential adverse effects are yet to be proven. This study aims to evaluate the association between calcium channel blockers exposure and the incidence of second primary malignancy. We established a cohort of 1401 patients with colorectal cancer diagnosed in our institution between January 2003 and December 2016. Patients were followed-up until December 2020. The tumor characteristics and basic clinical data including medication information were obtained from the hospital information system database. Second malignancy was detected in 301 patients (21.5%), and occurred in 27.8% of patients who used calcium channel blockers compared to only 19.9% among non-users. Their use was associated with an increased incidence of bladder cancer in particular. Subanalysis of patients with second malignancy displayed a higher proportion of right-sided colon cancer compared to rectal carcinoma in non-users. Survival analysis revealed significantly better outcomes in early-stage colorectal cancer patients without a history of calcium channel blockers treatment or second primary malignancy.
- MeSH
- blokátory kalciových kanálů MeSH
- kardiovaskulární nemoci * MeSH
- kolon MeSH
- lidé MeSH
- nádory rekta * MeSH
- nežádoucí účinky léčiv * MeSH
- sekundární malignity * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
