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BioRxiv : the Preprint Server For... Jun 2024Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are...
Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.
PubMed: 38915526
DOI: 10.1101/2024.06.12.598648 -
BioRxiv : the Preprint Server For... Jun 2024White matter hyperintensities (WMHs) are commonly detected on T2-weighted magnetic resonance imaging (MRI) scans, occurring in both typical aging and Alzheimer's...
White matter hyperintensities (WMHs) are commonly detected on T2-weighted magnetic resonance imaging (MRI) scans, occurring in both typical aging and Alzheimer's disease. Despite their frequent appearance and their association with cognitive decline, the molecular factors contributing to WMHs remain unclear. In this study, we investigated the transcriptomic profiles of two commonly affected brain regions with coincident AD pathology-frontal subcortical white matter (frontal-WM) and occipital subcortical white matter (occipital-WM)-and compared with age-matched healthy controls. Through RNA-sequencing in frontal- and occipital-WM bulk tissues, we identified an upregulation of genes associated with brain vasculature function in AD white matter. To further elucidate vasculature-specific transcriptomic features, we performed RNA-seq analysis on blood vessels isolated from these white matter regions, which revealed an upregulation of genes related to protein folding pathways. Finally, comparing gene expression profiles between AD individuals with high- versus low-WMH burden showed an increased expression of pathways associated with immune function. Taken together, our study characterizes the diverse molecular profiles of white matter changes in AD compared to normal aging and provides new mechanistic insights processes underlying AD-related WMHs.
PubMed: 38915516
DOI: 10.1101/2024.06.13.598845 -
[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 -
Archives of Biochemistry and Biophysics Jun 2024Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects...
Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1% (sum of relative quantities of each N-glycan)], hybrid (6, 11.9%), and complex (36, 55.0%) structures [bi- (13, 26.1%), tri- (16, 21.5%), and tetra-antennary (7, 7.4%)]. These included bisecting N-acetylglucosamine (33, 56.6%), mono- to tri-fucosylation (32, 53.3%), mono- to tri-α-galactosylation (16, 20.7%), and mono- to tetra-β-galactosylation (36, 58.5%). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3%), non-fucosylation (10, 13.6%), non-α-galactosylation (26, 46.2%), and non-β-galactosylation (6, 8.4%) were also identified. The activity (100%) of biALP was reduced to 37.3 ± 0.2% (by de-fucosylation), 32.7 ± 2.9% (by de-α-galactosylation), and 0.2 ± 0.2% (by de-β-galactosylation), comparable to inhibition by 10 to 10 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially β-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.
PubMed: 38914216
DOI: 10.1016/j.abb.2024.110069 -
Aging and Disease Jun 2024Brain insulin resistance has recently been described as a metabolic abnormality of brain glucose homeostasis that has been proven to downregulate insulin receptors, both... (Review)
Review
Brain insulin resistance has recently been described as a metabolic abnormality of brain glucose homeostasis that has been proven to downregulate insulin receptors, both in astrocytes and neurons, triggering a reduction in glucose uptake and glycogen synthesis. This condition may generate a mismatch between brain's energy reserve and expenditure, mainly during high metabolic demand, which could be involved in the chronification of migraine and, in the long run, at least in certain subsets of patients, in the prodromic phase of Alzheimer's disease, along a putative metabolic physiopathological continuum. Indeed, the persistent disruption of glucose homeostasis and energy supply to neurons may eventually impair protein folding, an energy-requiring process, promoting pathological changes in Alzheimer's disease, such as amyloid-β deposition and tau hyperphosphorylation. Hopefully, the "neuroenergetic hypothesis" presented herein will provide further insight on there being a conceivable metabolic bridge between chronic migraine and Alzheimer's disease, elucidating novel potential targets for the prophylactic treatment of both diseases.
PubMed: 38913047
DOI: 10.14336/AD.2024.0351 -
Heliyon Jun 2024Protein engineering mechanisms can be an efficient approach to enhance the biochemical properties of various biocatalysts. Immobilization of biocatalysts and the... (Review)
Review
Protein engineering mechanisms can be an efficient approach to enhance the biochemical properties of various biocatalysts. Immobilization of biocatalysts and the introduction of new-to-nature chemical reactivities are also possible through the same mechanism. Discovering new protocols that enhance the catalytic active protein that possesses novelty in terms of being stable, active, and, stereoselectivity with functions could be identified as essential areas in terms of concurrent bioorganic chemistry (synergistic relationship between organic chemistry and biochemistry in the context of enzyme engineering). However, with our current level of knowledge about protein folding and its correlation with protein conformation and activities, it is almost impossible to design proteins with specific biological and physical properties. Hence, contemporary protein engineering typically involves reprogramming existing enzymes by mutagenesis to generate new phenotypes with desired properties. These processes ensure that limitations of naturally occurring enzymes are not encountered. For example, researchers have engineered cellulases and hemicellulases to withstand harsh conditions encountered during biomass pretreatment, such as high temperatures and acidic environments. By enhancing the activity and robustness of these enzymes, biofuel production becomes more economically viable and environmentally sustainable. Recent trends in enzyme engineering have enabled the development of tailored biocatalysts for pharmaceutical applications. For instance, researchers have engineered enzymes such as cytochrome P450s and amine oxidases to catalyze challenging reactions involved in drug synthesis. In addition to conventional methods, there has been an increasing application of machine learning techniques to identify patterns in data. These patterns are then used to predict protein structures, enhance enzyme solubility, stability, and function, forecast substrate specificity, and assist in rational protein design. In this review, we discussed recent trends in enzyme engineering to optimize the biochemical properties of various biocatalysts. Using examples relevant to biotechnology in engineering enzymes, we try to expatiate the significance of enzyme engineering with how these methods could be applied to optimize the biochemical properties of a naturally occurring enzyme.
PubMed: 38912509
DOI: 10.1016/j.heliyon.2024.e32673 -
Cellular and Molecular Life Sciences :... Jun 2024N-methyladenosine (mA) is one of the most prevalent and conserved RNA modifications. It controls several biological processes, including the biogenesis and function of...
N-methyladenosine (mA) is one of the most prevalent and conserved RNA modifications. It controls several biological processes, including the biogenesis and function of circular RNAs (circRNAs), which are a class of covalently closed-single stranded RNAs. Several studies have revealed that proteotoxic stress response induction could be a relevant anticancer therapy in Acute Myeloid Leukemia (AML). Furthermore, a strong molecular interaction between the mA mRNA modification factors and the suppression of the proteotoxic stress response has emerged. Since the proteasome inhibition leading to the imbalance in protein homeostasis is strictly linked to the stress response induction, we investigated the role of Bortezomib (Btz) on mA regulation and in particular its impact on the modulation of mA-modified circRNAs expression. Here, we show that treating AML cells with Btz downregulated the expression of the mA regulator WTAP at translational level, mainly because of increased oxidative stress. Indeed, Btz treatment promoted oxidative stress, with ROS generation and HMOX-1 activation and administration of the reducing agent N-acetylcysteine restored WTAP expression. Additionally, we identified mA-modified circRNAs modulated by Btz treatment, including circHIPK3, which is implicated in protein folding and oxidative stress regulation. These results highlight the intricate molecular networks involved in oxidative and ER stress induction in AML cells following proteotoxic stress response, laying the groundwork for future therapeutic strategies targeting these pathways.
Topics: Humans; RNA, Circular; Leukemia, Myeloid, Acute; Adenosine; Oxidative Stress; Bortezomib; Cell Line, Tumor; Reactive Oxygen Species; RNA Splicing Factors; Cell Cycle Proteins; Neoplastic Stem Cells; Heme Oxygenase-1; Protein Serine-Threonine Kinases; Intracellular Signaling Peptides and Proteins
PubMed: 38909325
DOI: 10.1007/s00018-024-05299-9 -
Biochimica Et Biophysica Acta.... Jun 2024Iron‑sulfur (FeS) clusters are inorganic protein cofactors that perform essential functions in many physiological processes. Spectroscopic techniques have historically... (Review)
Review
Iron‑sulfur (FeS) clusters are inorganic protein cofactors that perform essential functions in many physiological processes. Spectroscopic techniques have historically been used to elucidate details of FeS cluster type, their assembly and transfer, and changes in redox and ligand binding properties. Structural probes of protein topology, complex formation, and conformational dynamics are also necessary to fully understand these FeS protein systems. Recent developments in mass spectrometry (MS) instrumentation and methods provide new tools to investigate FeS cluster and structural properties. With the unique advantage of sampling all species in a mixture, MS-based methods can be utilized as a powerful complementary approach to probe native dynamic heterogeneity, interrogate protein folding and unfolding equilibria, and provide extensive insight into protein binding partners within an entire proteome. Here, we highlight key advances in FeS protein studies made possible by MS methodology and contribute an outlook for its role in the field.
PubMed: 38908802
DOI: 10.1016/j.bbamcr.2024.119784 -
The Journal of Biological Chemistry Jun 2024Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with... (Review)
Review
Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to additionally play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease.
PubMed: 38908752
DOI: 10.1016/j.jbc.2024.107488 -
Molecular Cell Jun 2024Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote...
Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
PubMed: 38908370
DOI: 10.1016/j.molcel.2024.06.002