-
The Journal of Biological Chemistry Jan 2024Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an...
Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.
Topics: Bone Morphogenetic Proteins; Ligands; Signal Transduction; Cell Line; Peptide Hormones; Protein Multimerization; Mutation; Recombinant Proteins; Protein Domains; Humans
PubMed: 37949218
DOI: 10.1016/j.jbc.2023.105452 -
Acta Tropica Jun 2024Cervids are highly exposed to ticks, however, their role in the life cycle of these rickettsiae has not been fully elucidated. Given the expanding distribution and...
Cervids are highly exposed to ticks, however, their role in the life cycle of these rickettsiae has not been fully elucidated. Given the expanding distribution and growing population of deer species in Portugal, coupled with their direct and indirect interactions with humans during hunting, it becomes crucial to explore their role as sentinels and potential reservoirs of Rickettsia. The present investigation aimed to detect and evaluate exposure to Rickettsia in free-living deer from Portugal. Blood samples (n = 77) were collected from hunted game animals (red deer and fallow deer) from different areas throughout Portugal (Idanha-a-Nova, Monte Fidalgo, Montalvão and Arraiolos) and sera were tested by immunofluorescence assay, to detect antibodies. Additionally, blood DNA samples were screened for SFGR by nested-polymerase chain reaction targeting a fragment of the outer membrane protein B (ompB) gene, as well as for Anaplasma and Ehrlichia spp. targeting the 16S rRNA gene. Thirty-five per cent (25 deer and two fallow deer) tested positive (sera with a titer ≥1:64) for IgG antibodies against Rickettsia conorii. No rickettsial DNA was detected by PCR for the ompB gene, and all DNA samples tested negative for Anaplasma and Ehrlichia. As far as we know, this study is the first screening of cervid species in Portugal for Rickettsia antibodies. The findings suggest that these animals serve as useful sentinel indicators for the circulation of rickettsiae, offering a complementary perspective to studies focused on ticks. The increasing numbers of hunted deer in Portugal and the potential zoonotic features of Rickettsia spp. highlight the importance of continued surveillance directed at tick-borne diseases, especially those involving wild animals.
Topics: Animals; Portugal; Deer; Antibodies, Bacterial; Rickettsia; Rickettsia Infections; Sentinel Species; DNA, Bacterial; Immunoglobulin G; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Anaplasma; Ehrlichia; Rickettsia conorii; Bacterial Outer Membrane Proteins; Male
PubMed: 38565332
DOI: 10.1016/j.actatropica.2024.107202 -
Gut Microbes 2024Colorectal cancer (CRC), a malignant tumor worldwide, is associated with gut microbiota. The influence of gut microbe-derived metabolites on CRC has attracted a lot of...
Colorectal cancer (CRC), a malignant tumor worldwide, is associated with gut microbiota. The influence of gut microbe-derived metabolites on CRC has attracted a lot of attention. However, the role of immunity mediated by commensal microbiota-derived metabolites in tumorigenesis of CRC is not intensively explored. Here we monitored the gut microbial dysbiosis in CRC mouse model ( model) without dietary and pharmacological intervention, followed by characterized of metabolites enriched in CRC model mice. Profound changes of gut microbiome (bacteriome) were observed during intestinal disorders. Metabolomic profiling indicated that agmatine, derived from the gut bacteria and , could interact with Rnf128 to suppress the Rnf128-mediated ubiquitination of β-catenin to further upregulate the downstream targets of β-catenin including Cyclin D1, Lgr5, CD44 and C-myc, thus activating Wnt signaling. The activated Wnt signaling pathway promoted dysplasia of intestinal cells and inflammatory infiltration of lymphocytes via inducing the upregulation of pro-inflammatory cytokines (IL-6 and TNF-α) and downregulation of anti-inflammatory cytokine (IL-10), thereby contributing to colorectal carcinogenesis. Therefore, our study presented novel insights into the roles and mechanisms of gut microbiota in pathogenesis of CRC.
Topics: Animals; Gastrointestinal Microbiome; Colorectal Neoplasms; Mice; Carcinogenesis; Wnt Signaling Pathway; Inflammation; Bacteria; Mice, Inbred C57BL; beta Catenin; Dysbiosis; Humans; Disease Models, Animal; Cytokines; Symbiosis; Male
PubMed: 38706224
DOI: 10.1080/19490976.2024.2348441 -
International Journal of Molecular... May 2024Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and...
Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 , lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.
Topics: alpha-Synuclein; Humans; Parkinson Disease; Osmolar Concentration; Reproducibility of Results; Escherichia coli
PubMed: 38892177
DOI: 10.3390/ijms25115988 -
Drug Design, Development and Therapy 2024In an era where synthetic supplements have raised concerns regarding their effects on human health, has emerged as a natural alternative rich in polyphenolic compounds... (Review)
Review
In an era where synthetic supplements have raised concerns regarding their effects on human health, has emerged as a natural alternative rich in polyphenolic compounds with potent therapeutic properties. Various studies on focusing on the analysis and validation of its pharmacological and nutritional properties are emerging. This paper summarizes present data and information on the phytochemical, nutritional values, therapeutic potential, as well as the toxicity profile of . An extensive search was conducted from various databases, including PubMed, ScienceDirect, Scopus, and Google Scholar. A total of 126 studies and articles related to that were published between 1999 and 2023 were included in this review. Remarkably, exhibits a diverse array of advantageous effects, including, but not limited to, antioxidant, anti-neurodegenerative, antimicrobial, antiviral, anti-inflammatory, anti-arthritic, antiepileptic, anticonvulsant, anti-hyperlipidemic, anti-angiogenic, antidiabetic, anti-cancer, and antimutagenic properties. Among the highlights include that antioxidants from were demonstrated to inhibit cholinesterase, potentially protecting neurons in Alzheimer's disease and other neurodegenerative conditions. The antimicrobial activities of were attributed to its high flavonoids and terpenoids content, while its virucidal action through the inhibition of DNA and RNA replication was postulated due to its triterpenes content. Inflammatory and arthritic conditions may also benefit from its anti-inflammatory and anti-arthritic properties through the modulation of various signalling proteins. Studies have also shown that extracts were generally safe and exhibit low toxicity profile, although more research in this aspect is required, specifically its effects on the skin. In conclusion, this study highlights the potential of as a valuable natural therapeutic agent and dietary supplement. However, continued exploration on s safety and efficacy is still required prior to embarking on clinical trials, as its role in personalized nutrition and medication will open a new paradigm to improve health outcomes.
Topics: Ficus; Humans; Dietary Supplements; Animals; Plant Extracts; Antioxidants; Phytochemicals
PubMed: 38831870
DOI: 10.2147/DDDT.S436446 -
Cells Jul 2023A phenotypic hallmark of cancer is aberrant transcriptional regulation. Transcriptional regulation is controlled by a complicated array of molecular factors, including... (Review)
Review
A phenotypic hallmark of cancer is aberrant transcriptional regulation. Transcriptional regulation is controlled by a complicated array of molecular factors, including the presence of transcription factors, the deposition of histone post-translational modifications, and long-range DNA interactions. Determining the molecular identity and function of these various factors is necessary to understand specific aspects of cancer biology and reveal potential therapeutic targets. Regulation of the genome by specific factors is typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) that identifies genome-wide binding interactions through the use of factor-specific antibodies. A long-standing goal in many laboratories has been the development of a 'reverse-ChIP' approach to identify unknown binding partners at loci of interest. A variety of strategies have been employed to enable the selective biochemical purification of sequence-defined chromatin regions, including single-copy loci, and the subsequent analytical detection of associated proteins. This review covers mass spectrometry techniques that enable quantitative proteomics before providing a survey of approaches toward the development of strategies for the purification of sequence-specific chromatin as a 'reverse-ChIP' technique. A fully realized reverse-ChIP technique holds great potential for identifying cancer-specific targets and the development of personalized therapeutic regimens.
Topics: Humans; Proteome; Chromatin; DNA; Histones; Neoplasms
PubMed: 37508524
DOI: 10.3390/cells12141860 -
Emerging Microbes & Infections Dec 2023Epidemiological characteristics and molecular features of carbapenem-resistant (CR-) species remain unclear in China. In this study, we performed a genomic study on 92...
Epidemiological characteristics and molecular features of carbapenem-resistant (CR-) species remain unclear in China. In this study, we performed a genomic study on 92 isolates from -caused infections from a multicenter study in China. Whole genome sequencing (WGS) was used to determine the genome sequence of 92 non-duplicated CR- strains collected from multiple tertiary health centres. The precise species of strains were identified by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH). Molecular features of high-risk CR- sequence type (ST) lineages and carbapenemase-encoding plasmids were determined. The result revealed that the most common human-source CR- species in China was (66/92, 71.93%), and the proportion of carbapenemase-producing (CP-) in CR- was high (72/92, 78.26%) in comparison to other global regions. Furthermore, ST171 and ST116 were the major lineages of CP- strains, and ST171 was more likely to cause infections in older patients. Genomic analysis also highlighted the likelihood of intra-hospital/inter-hospital clonal transmission of ST171 and ST116 . In addition, the -harbouring IncX3-type plasmid was identified as the prevalent carbapenemase-encoding plasmid carried by CR- strains, and was experimentally confirmed to be able to self-transfer with high frequency. This study detailed the genomic and clinical characteristics of CR- in China in the form of multicenter for the first time. The high risk of carbapenemase-producing ST171 and ST116 , and the -harbouring IncX3-type plasmid were detected and emphasized.
Topics: Aged; Humans; Bacterial Proteins; beta-Lactamases; Carbapenem-Resistant Enterobacteriaceae; China; Enterobacter; Enterobacteriaceae Infections; Genomics; Microbial Sensitivity Tests; Plasmids
PubMed: 36382635
DOI: 10.1080/22221751.2022.2148562 -
Se Pu = Chinese Journal of... Jun 2024Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability,... (Review)
Review
Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.
Topics: Antibodies; Antibodies, Monoclonal; Chromatography; Chromatography, Affinity; Chromatography, Ion Exchange; Hydrophobic and Hydrophilic Interactions
PubMed: 38845514
DOI: 10.3724/SP.J.1123.2023.12010 -
Molecular & Cellular Proteomics : MCP Dec 2023Proteins can be modified by lipids in various ways, for example, by myristoylation, palmitoylation, farnesylation, and geranylgeranylation-these processes are...
Proteins can be modified by lipids in various ways, for example, by myristoylation, palmitoylation, farnesylation, and geranylgeranylation-these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analysis of endogenous protein N-terminal myristoylation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore myristoylation sites in HeLa cells and identified a total of 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence myristoylated proteins identified by myristic acid analog-based chemical proteomics. Isolation of myristoylated peptides from HeLa digests prepared with different proteases enabled the identification of different myristoylated sites, extending the coverage of N-myristoylome. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of protein N-terminal myristoylation.
Topics: Humans; Animals; Mice; Myristic Acid; HeLa Cells; Proteomics; Proteins; Peptides; Liquid-Liquid Extraction; Protein Processing, Post-Translational
PubMed: 37949301
DOI: 10.1016/j.mcpro.2023.100677 -
The Journal of Biological Chemistry Jul 2023Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH...
Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 10 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a K of <80 nM, a T of >70 °C, and a t in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.
Topics: Antibodies; Chromatography, Affinity; Ligands; Protein Engineering; Recombinant Proteins; Sodium Hydroxide; Antibody Affinity; Molecular Mimicry; Protein Stability; Hot Temperature; Trypsin; Recombinant Fusion Proteins; Protein Binding
PubMed: 37315789
DOI: 10.1016/j.jbc.2023.104910