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Molecules (Basel, Switzerland) Feb 2024Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. is one of the most common...
Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors-4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated.
Topics: Humans; Blood Vessel Prosthesis; Anti-Infective Agents; Anti-Bacterial Agents; Biofilms; Serine Proteinase Inhibitors; Staphylococcus aureus; Staphylococcal Infections; Biocompatible Materials; Puromycin; Peptide Hydrolases; Sulfones
PubMed: 38474448
DOI: 10.3390/molecules29050935 -
Scientific Reports Mar 2024Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB...
Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.
Topics: Humans; Shiga Toxins; Escherichia coli; Cell-Free System; HeLa Cells; Protein Biosynthesis
PubMed: 38472311
DOI: 10.1038/s41598-024-56190-3 -
BioRxiv : the Preprint Server For... Jan 2024As microtubule-organizing centers, centrosomes direct assembly of the bipolar mitotic spindle required for chromosome segregation and genome stability. Centrosome...
As microtubule-organizing centers, centrosomes direct assembly of the bipolar mitotic spindle required for chromosome segregation and genome stability. Centrosome activity requires the dynamic assembly of pericentriolar material (PCM), the composition and organization of which changes throughout the cell cycle. Recent studies highlight the conserved localization of several mRNAs encoded from centrosome-associated genes enriched at centrosomes, including () mRNA. However, relatively little is known about how RNAs localize to centrosomes and influence centrosome function. Here, we examine mechanisms underlying the subcellular localization of mRNA. We find that mRNA localization is puromycin-sensitive, and the coding sequence is both necessary and sufficient for RNA localization, consistent with a co-translational transport mechanism. We identify regions within the coding sequence that regulate mRNA localization. Finally, we show that protein-protein interactions critical for elaboration of the PCM scaffold permit RNA localization to centrosomes. Taken together, these findings inform the mechanistic basis of mRNA localization and lend insight into the oscillatory enrichment of RNA at centrosomes.
PubMed: 38469150
DOI: 10.1101/2024.01.13.575509 -
Bio-protocol Mar 2024The parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The...
The parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the -HepG2 infection model, followed by automated confocal image acquisition and computational separation of vs. nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis. Key features • Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in liver-stage parasites in actively translating hepatoma cells. • This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells. • The -infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification.
PubMed: 38464937
DOI: 10.21769/BioProtoc.4952 -
BioRxiv : the Preprint Server For... Mar 2024Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria...
Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.
PubMed: 38464017
DOI: 10.1101/2023.06.20.544360 -
STAR Protocols Mar 2024Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX...
Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression. For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al..
Topics: Humans; CRISPR-Cas Systems; Induced Pluripotent Stem Cells; Neurodegenerative Diseases; Gene Editing; Macrophages
PubMed: 38401123
DOI: 10.1016/j.xpro.2024.102903 -
The Journal of Biological Chemistry Mar 2024Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and...
Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.
Topics: Escherichia coli; Escherichia coli Proteins; Peptides; Protein Biosynthesis; Puromycin; Ribosomes
PubMed: 38395310
DOI: 10.1016/j.jbc.2024.105780 -
Bio-protocol Feb 2024As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to...
As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to the cellular microenvironment. Consequently, variations in protein synthesis rates occur under various physiological and pathological conditions, enabling an adaptive response by the cell. For example, global protein synthesis increases upon mitogenic factors to support biomass generation and cell proliferation, while exposure to low concentrations of oxygen or nutrients require translational repression and reprogramming to avoid energy depletion and cell death. To assess fluctuations in protein synthesis rates, radioactive isotopes or radiolabeled amino acids are often used. Although highly sensitive, these techniques involve the use of potentially toxic radioactive compounds and require specific materials and processes for the use and disposal of these molecules. The development of alternative, non-radioactive methods that can be easily and safely implemented in laboratories has therefore been encouraged to avoid handling radioactivity. In this context, the SUrface SEnsing of Translation (SUnSET) method, based on the classical western blot technique, was developed by Schmidt et al. in 2009. The SUnSET is nowadays recognized as a simple alternative to radioactive methods assessing protein synthesis rates. Key features • As a structural analogue of aminoacyl-transfer RNA, puromycin incorporates into the elongating peptide chain. • Detection of puromycin-labeled peptides by western blotting reflects translation rates without the need for radioactive isotopes. • The protocol described here for in vitro applications is derived from the SUnSET method originally published by Schmidt et al. (2009).
PubMed: 38379826
DOI: 10.21769/BioProtoc.4933 -
BioRxiv : the Preprint Server For... Feb 2024Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and...
Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal non-canonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not PABPC1, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of the putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes and can stimulate NGD of a select set of transcripts in cells, revealing an unappreciated role of FMRP that would be misregulated in FXS.
PubMed: 38352534
DOI: 10.1101/2024.02.02.577121 -
International Journal of Molecular... Jan 2024Vaccinia virus () F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected...
Vaccinia virus () F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17 particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17 particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17 particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17 particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17 particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17 particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17 particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17 particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.
Topics: Humans; Vaccinia virus; Vaccinia; Isopropyl Thiogalactoside; Cell Line; Phosphoproteins; Virion
PubMed: 38338659
DOI: 10.3390/ijms25031382