-
Non-coding RNA Jun 2024Telomerase is an enzyme involved in the maintenance of telomeres. Telomere shortening due to the end-replication problem is a threat to the genome integrity of all... (Review)
Review
Telomerase is an enzyme involved in the maintenance of telomeres. Telomere shortening due to the end-replication problem is a threat to the genome integrity of all eukaryotes. Telomerase inside cells depends on a myriad of protein-protein and RNA-protein interactions to properly assemble and regulate the function of the telomerase holoenzyme. These interactions are well studied in model eukaryotes, like humans, yeast, and the ciliated protozoan known as . Emerging evidence also suggests that deep-branching eukaryotes, such as the parasitic protist require conserved and novel RNA-binding proteins for the assembly and function of their telomerase. In this review, we will discuss telomerase regulatory pathways in the context of telomerase-interacting proteins, with special attention paid to RNA-binding proteins. We will discuss these interactors on an evolutionary scale, from parasitic protists to humans, to provide a broader perspective on the extensive role that protein-protein and RNA-protein interactions play in regulating telomerase activity in eukaryotes.
PubMed: 38921833
DOI: 10.3390/ncrna10030036 -
Cells Jun 2024The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the...
The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.
Topics: Animals; Cullin Proteins; Dendrites; Drosophila melanogaster; Drosophila Proteins; Huntingtin Protein; Larva; Mechanistic Target of Rapamycin Complex 1; Microtubules; Sensory Receptor Cells; Signal Transduction; Transcription Factors; Chaperonin Containing TCP-1
PubMed: 38920658
DOI: 10.3390/cells13121029 -
Cells Jun 2024Neurodegenerative disorders are affecting millions of people worldwide, impacting the healthcare system of our society. Among them, Alzheimer's disease (AD) is the most...
Neurodegenerative disorders are affecting millions of people worldwide, impacting the healthcare system of our society. Among them, Alzheimer's disease (AD) is the most common form of dementia, characterized by severe cognitive impairments. Neuropathological hallmarks of AD are β-amyloid (Aβ) plaques and neurofibrillary tangles, as well as endoplasmic reticulum and mitochondria dysfunctions, which finally lead to apoptosis and neuronal loss. Since, to date, there is no definitive cure, new therapeutic and prevention strategies are of crucial importance. In this scenario, cannabinoids are deeply investigated as promising neuroprotective compounds for AD. In this study, we evaluated the potential neuroprotective role of cannabinerol (CBNR) in an in vitro cellular model of AD via next-generation sequencing. We observed that CBNR pretreatment counteracts the Aβ-induced loss of cell viability of differentiated SH-SY5Y cells. Moreover, a network-based transcriptomic analysis revealed that CBNR restores normal mitochondrial and endoplasmic reticulum functions in the AD model. Specifically, the most important genes regulated by CBNR are related mainly to oxidative phosphorylation (, , , ), protein folding () and degradation (, , ), and glucose () and lipid (, , ) metabolism. Therefore, these results suggest that CBNR could be a new neuroprotective agent helpful in the prevention of AD dysfunctions.
Topics: Humans; Alzheimer Disease; Mitochondria; Endoplasmic Reticulum; Cannabinoids; Amyloid beta-Peptides; Endoplasmic Reticulum Chaperone BiP; Cell Line, Tumor; Gene Expression Profiling; Transcriptome; Cell Survival; Neuroprotective Agents; Models, Biological; Gene Regulatory Networks
PubMed: 38920643
DOI: 10.3390/cells13121012 -
Frontiers in Microbiology 2024In recent years, there has been an increasing tendency to create drugs based on certain commensal bacteria of the human microbiota and their ingredients, primarily...
In recent years, there has been an increasing tendency to create drugs based on certain commensal bacteria of the human microbiota and their ingredients, primarily focusing on live biotherapeutics (LBPs) and postbiotics. The creation of such drugs, termed pharmacobiotics, necessitates an understanding of their mechanisms of action and the identification of pharmacologically active ingredients that determine their target properties. Typically, these are complexes of biologically active substances synthesized by specific strains, promoted as LBPs or postbiotics (including vesicles): proteins, enzymes, low molecular weight metabolites, small RNAs, etc. This study employs omics technologies, including genomics, proteomics, and metabolomics, to explore the potential of U-21 for innovative LBP and postbiotic formulations targeting neuroinflammatory processes. Proteomic techniques identified and quantified proteins expressed by U-21, highlighting their functional attributes and potential applications. Key identified proteins include ATP-dependent Clp protease (ClpL), chaperone protein DnaK, protein GrpE, thioredoxin reductase, LysM peptidoglycan-binding domain-containing protein, and NlpC/P60 domain-containing protein, which have roles in disaggregase, antioxidant, and immunomodulatory activities. Metabolomic analysis provided insights into small-molecule metabolites produced during fermentation, revealing compounds with anti-neuroinflammatory activity. Significant metabolites produced by U-21 include GABA (γ-aminobutyric acid), niacin, aucubin, and scyllo-inositol. GABA was found to stabilize neuronal activity, potentially counteracting neurodegenerative processes. Niacin, essential for optimal nervous system function, was detected in vesicles and culture fluid, and it modulates cytokine production, maintaining immune homeostasis. Aucubin, an iridoid glycoside usually secreted by plants, was identified as having antioxidant properties, addressing issues of bioavailability for therapeutic use. Scyllo-inositol, identified in vesicles, acts as a chemical chaperone, reducing abnormal protein clumps linked to neurodegenerative diseases. These findings demonstrate the capability of U-21 to produce bioactive substances that could be harnessed in the development of pharmacobiotics for neurodegenerative diseases, contributing to their immunomodulatory, anti-neuroinflammatory, and neuromodulatory activities. Data of the HPLC-MS/MS analysis are available via ProteomeXchange with identifier PXD050857.
PubMed: 38919499
DOI: 10.3389/fmicb.2024.1416688 -
Open Biology Jun 2024is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S]...
is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including . [2Fe-2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe-2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.
Topics: Aspergillus fumigatus; Fungal Proteins; Cytosol; Mitochondria; Iron; Adaptation, Physiological; Cell Nucleus; Protein Transport; Proteomics; Iron-Sulfur Proteins; Gene Expression Regulation, Fungal; Acetylation
PubMed: 38919062
DOI: 10.1098/rsob.240033 -
PloS One 2024SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at...
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.
Topics: Escherichia coli Proteins; Peptidylprolyl Isomerase; Escherichia coli; Protein Binding; Molecular Chaperones; Protein Sorting Signals; Hydrophobic and Hydrophilic Interactions; Calorimetry; Arginine; Amino Acid Sequence
PubMed: 38917203
DOI: 10.1371/journal.pone.0305823 -
Microbiology Spectrum Jun 2024Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with...
UNLABELLED
Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with (), these processes are essential for host-pathogen interactions and immune responses. However, the specific effects of acetylation and deacetylation on cellular functions during infection are not fully understood. This study employed Tandem Mass Tag (TMT) labeling for quantitative proteomic profiling to examine the acetylproteome (acetylome) profiles of noninfected and -infected macrophages. We identified 715 acetylated peptides from 1,072 proteins and quantified 544 lysine acetylation sites (Kac) in 402 proteins in noninfected and -infected macrophages. Our research revealed a link between acetylation events and metabolic changes during infection. Notably, the deacetylation of heat shock protein 60 (HSP60), a key chaperone protein, was significantly associated with this process. Specifically, the deacetylation of HSP60 at K96 by sirtuin3 (SIRT3) enhances macrophage apoptosis, leading to the elimination of intracellular . These findings underscore the pivotal role of the SIRT3-HSP60 axis in the host immune response to . This study offers a new perspective on host protein acetylation and suggests that targeting host-directed therapies could be a promising approach for tuberculosis immunotherapy.
IMPORTANCE
Protein acetylation is crucial for the onset, development, and outcome of tuberculosis (TB). Our study comprehensively investigated the dynamics of lysine acetylation during infection, shedding light on the intricate host-pathogen interactions that underlie the pathogenesis of tuberculosis. Using an advanced quantitative lysine proteomics approach, different profiles of acetylation sites and proteins in macrophages infected with were identified. Functional enrichment and protein-protein network analyses revealed significant associations between acetylated proteins and key cellular pathways, highlighting their critical role in the host response to infection. Furthermore, the deacetylation of HSP60 and its influence on macrophage-mediated clearance of underscore the functional significance of acetylation in tuberculosis pathogenesis. In conclusion, this study provides valuable insights into the regulatory mechanisms governing host immune responses to infection and offers promising avenues for developing novel therapeutic interventions against TB.
PubMed: 38916288
DOI: 10.1128/spectrum.00749-24 -
[Analysis of enzyme activity and substrate specificity of dolichyl-phosphate β-glucosyltransferase].Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2024Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent...
Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated -glycan moieties on peptide. It is of great significance to study the -glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues . In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate β-glucosyltransferase E (Alg5E) derived from . The results demonstrated that the recombinant Alg5E, expressed in , exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).
Topics: Glucosyltransferases; Substrate Specificity; Escherichia coli; Trichomonas vaginalis; Recombinant Proteins; Dolichol Phosphates; Endoplasmic Reticulum
PubMed: 38914494
DOI: 10.13345/j.cjb.230737 -
Journal of Applied Biomedicine Jun 2024Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory...
Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory activity of RSV in traumatic brain injury (TBI) in mice associated with endoplasmic reticulum stress (ERS). After establishing three experimental groups (sham, TBI, and TBI+RSV), we explored the results of RSV after TBI on ERS and caspase-12 apoptotic pathways. The expression levels of C/EBP homologous protein (CHOP), glucose regulated protein 78kD (GRP78), caspase-3, and caspase-12 in cortical brain tissues were assessed by western blotting. The qPCR analysis was also performed on mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in cortical brain tissue. In addition, the expression of GRP78 in microglia (ionized calcium binding adaptor molecule 1; Iba-1) and neurons (neuronal nuclei; NeuN) was identified by immunofluorescence staining. The neurological function of mice was assessed by modified neurological severity scores (mNSS). After drug treatment, the expression of CHOP, GRP78, caspase-3 and caspase-12 decreased, and qPCR results showed that TNF-α and IL-1β were down-regulated. Immunofluorescence staining showed down-regulation of Iba-1+/GRP78+ and NeuN+/GRP78+ cells after RSV treatment. The mNSS analysis confirmed improvement after RSV treatment. RSV improved apoptosis by downregulating the ERS signaling pathway and improved neurological prognosis in mice with TBI.
Topics: Animals; Brain Injuries, Traumatic; Resveratrol; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Mice; Male; Apoptosis; Prognosis; Neuroprotective Agents; Neurons; Interleukin-1beta; Caspase 12; Heat-Shock Proteins; Tumor Necrosis Factor-alpha; Mice, Inbred C57BL; Cell Death; Microglia; Transcription Factor CHOP
PubMed: 38912865
DOI: 10.32725/jab.2024.008 -
JCI Insight Jun 2024Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase...
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Topics: Animals; Male; Spermatogenesis; Protein Disulfide-Isomerases; Mice; Mice, Knockout; Testis; Endoplasmic Reticulum Chaperone BiP; Infertility, Male; Apoptosis; Spermatocytes; Endoplasmic Reticulum Stress; Oligospermia
PubMed: 38912589
DOI: 10.1172/jci.insight.177743