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Experimental Parasitology Aug 2023Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various...
Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 μM, and for ATP 291 ± 15 and 38 ± 2.2 μM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.
Topics: Humans; Trypanosoma cruzi; Phosphoglycerate Kinase; Protein Isoforms; Chagas Disease; Adenosine Triphosphate
PubMed: 37353138
DOI: 10.1016/j.exppara.2023.108574 -
The Journal of Biological Chemistry Jul 2023A common feature among nearly all gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides...
A common feature among nearly all gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane, which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate (CPG). Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg as a cofactor. Mn, but no other divalent cations, could substitute for Mg. Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD C6-ceramide (K = 19.2 ± 5.5 μM; V = 2590 ± 230 pmol/min/mg enzyme) and ATP (K = 0.29 ± 0.07 mM; V = 10,100 ± 996 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases, which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.
Topics: Humans; Caulobacter crescentus; Ceramides; Lipopolysaccharides; Phylogeny; Sphingolipids
PubMed: 37286040
DOI: 10.1016/j.jbc.2023.104894 -
Nature Communications Feb 2024The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of...
The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of downregulating EGFR function involves its endocytic transport to the lysosome. Sorting of proteins into intracellular pathways involves cargo adaptors recognizing sorting signals on cargo proteins. A dileucine-based sorting signal has been identified previously for the sorting of endosomal EGFR to the lysosome, but a cargo adaptor that recognizes this signal remains unknown. Here, we find that phosphoglycerate kinase 1 (PGK1) is recruited to endosomal membrane upon its phosphorylation, where it binds to the dileucine sorting signal in EGFR to promote the lysosomal transport of this receptor. We also elucidate two mechanisms that act in concert to promote PGK1 recruitment to endosomal membrane, a lipid-based mechanism that involves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and a protein-based mechanism that involves hepatocyte growth factor receptor substrate (Hrs). These findings reveal an unexpected function for a metabolic enzyme and advance the mechanistic understanding of how EGFR is transported to the lysosome.
Topics: Phosphoglycerate Kinase; ErbB Receptors; Endosomes; Proteins; Lysosomes; Protein Transport; Endosomal Sorting Complexes Required for Transport
PubMed: 38310114
DOI: 10.1038/s41467-024-45443-4 -
Gene Therapy Dec 2023Spinal muscular atrophy (SMA) is a neuromuscular disease particularly characterised by degeneration of ventral motor neurons. Survival motor neuron (SMN) 1 gene...
Spinal muscular atrophy (SMA) is a neuromuscular disease particularly characterised by degeneration of ventral motor neurons. Survival motor neuron (SMN) 1 gene mutations cause SMA, and gene addition strategies to replace the faulty SMN1 copy are a therapeutic option. We have developed a novel, codon-optimised hSMN1 transgene and produced integration-proficient and integration-deficient lentiviral vectors with cytomegalovirus (CMV), human synapsin (hSYN) or human phosphoglycerate kinase (hPGK) promoters to determine the optimal expression cassette configuration. Integrating, CMV-driven and codon-optimised hSMN1 lentiviral vectors resulted in the highest production of functional SMN protein in vitro. Integration-deficient lentiviral vectors also led to significant expression of the optimised transgene and are expected to be safer than integrating vectors. Lentiviral delivery in culture led to activation of the DNA damage response, in particular elevating levels of phosphorylated ataxia telangiectasia mutated (pATM) and γH2AX, but the optimised hSMN1 transgene showed some protective effects. Neonatal delivery of adeno-associated viral vector (AAV9) vector encoding the optimised transgene to the Smn mouse model of SMA resulted in a significant increase of SMN protein levels in liver and spinal cord. This work shows the potential of a novel codon-optimised hSMN1 transgene as a therapeutic strategy for SMA.
Topics: Animals; Humans; Infant, Newborn; Mice; Cytomegalovirus Infections; Disease Models, Animal; DNA, Complementary; Motor Neurons; Muscular Atrophy, Spinal; Survival of Motor Neuron 1 Protein; Transcription Factors; Transgenes
PubMed: 37322133
DOI: 10.1038/s41434-023-00406-0 -
Journal of Cancer Research and Clinical... Sep 2023Early diagnosis is crucial for optimal prognosis of gastric cancer (GC). Hereby, we aimed to identify novel serum autoantibody-based biomarkers for precancerous lesion...
PURPOSE
Early diagnosis is crucial for optimal prognosis of gastric cancer (GC). Hereby, we aimed to identify novel serum autoantibody-based biomarkers for precancerous lesion (PL) and early GC.
METHODS
We performed serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (Nano-LC-Q-TOF-MS/MS) to screen for GC-associated autoantibodies. The identified autoantibodies were analyzed for potential detection value for PL and GC by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves analysis was conducted to evaluate the accuracy of the biomarkers.
RESULTS
We identified seven candidates, such as mRNA export factor (RAE1), Nucleophosmin 1 (NPM1), phosphoglycerate kinase 1 (PGK1), and ADP-ribosylation factor 4 (ARF4). Antibodies against all seven proteins were present at higher levels in sera from 242 patients (51 PL, 78 early GC, 113 advanced GC) compared with sera from 122 healthy individuals. RAE1-specific autoantibody discriminated best between patients at different GC stages, with area under the curve (AUC) values of 0.710, 0.745, and 0.804 for PL, early GC, and advanced GC, respectively. Two predictive models composed of gender, RAE1, PGK1, NPM1, and ARF4 autoantibodies (Model 2 for PL) and of age, gender, RAE1, PGK1, and NPM1 autoantibodies (Model 3 for early GC) had improved diagnostic efficiencies, with AUCs of 0.803 and 0.857, sensitivities of 66.7% and 75.6%, and specificities of 78.7% and 87.7%, respectively.
CONCLUSION
The identified serum tumor-associated autoantibodies (TAAbs) may have good potential for early detection of GC and PL.
Topics: Humans; Biomarkers, Tumor; Autoantibodies; Stomach Neoplasms; Tandem Mass Spectrometry; ROC Curve; Early Detection of Cancer; Nuclear Proteins; Precancerous Conditions
PubMed: 37079049
DOI: 10.1007/s00432-023-04732-z -
CNS Neuroscience & Therapeutics Apr 2024Despite extensive work to identify diagnostic plasma markers for Parkinson's disease (PD), there are still no accepted and validated surrogate biomarkers....
BACKGROUND
Despite extensive work to identify diagnostic plasma markers for Parkinson's disease (PD), there are still no accepted and validated surrogate biomarkers. Mitophagy-associated proteins (MAPs), including PTEN-induced putative kinase 1 (PINK1), Parkin, phosphoglycerate mutase 5 (PGAM5), BCL2 interacting protein 3 (BNIP3), and phosphorylated-TBK1 (p-TBK1), are, to our best knowledge, not well studied as a panel of biomarkers of neurodegeneration in PD.
METHODS
The study population comprised 116 age-matched controls (HC), 179 PD patients, alongside and 90 PD syndromes (PDs) divided between two cohorts: (i) the modeling cohort (cohort 1), including 150 PD, 97 HC, and 80 PDs; and (ii) the validated cohort (cohort 2), including 29 PD, 19 HC, and 10 PDs.
RESULTS
MAPs are elevated in the plasma of PD patients. PINK1, Parkin, and PGAM5 displayed the top three measurable increase trends in amplitude compared to BNIP3 and p-TBK1. Moreover, the area under the curve (AUC) values of PINK1, PGAM5, and Parkin were ranked the top three MAP candidates in diagnosis accuracy for PD from HC, but the MAPs make it hard to differentiate PD from PDs. In addition, there are higher plasma PINK1-Parkin levels and prominent diagnostic accuracy in A-synuclein (+) subjects than in A-synuclein (-) subjects.
CONCLUSIONS
These results uncover that plasma MAPs (PINK1, Parkin, and PGAM5) may be potentially useful diagnostic biomarkers for PD diagnosis. Studies on larger cohorts would be required to test whether elevated plasma MAP levels are related to PD risk or prognosis.
Topics: Humans; Parkinson Disease; Male; Female; Biomarkers; Aged; Middle Aged; Mitophagy; Protein Kinases; Ubiquitin-Protein Ligases; Cohort Studies; Mitochondrial Proteins; Membrane Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Phosphoprotein Phosphatases
PubMed: 37990436
DOI: 10.1111/cns.14532 -
Proceedings of the National Academy of... Feb 2024The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding...
The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.
Topics: Humans; Animals; Phosphoglycerate Kinase; Prazosin; Parkinson Disease; Glycolysis
PubMed: 38377207
DOI: 10.1073/pnas.2318956121 -
Biochemical and Biophysical Research... Jul 2023Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the...
Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.
Topics: Phosphorylation; Protein Kinases; Bacillus anthracis; Phosphoglycerate Mutase; Threonine; Spores, Bacterial; Bacterial Proteins
PubMed: 37149987
DOI: 10.1016/j.bbrc.2023.04.039 -
International Journal of Molecular... Jan 2024Aberrant protein post-translational modification is a hallmark of malignant tumors. Lysine succinylation (Ksucc) plays a vital role in cell energy metabolism in various...
Aberrant protein post-translational modification is a hallmark of malignant tumors. Lysine succinylation (Ksucc) plays a vital role in cell energy metabolism in various cancers. However, whether succinylation can be catalyzed by acetyltransferase p300 remains unclear. In this study, we unveiled that p300 is a "writer" for succinylation, and p300-mediated Ksucc promotes cell glycometabolism in lung adenocarcinoma (LUAD). Specifically, our succinylome data revealed that deficiency leads to the systemic reduction of Ksucc, and 79.55% of the p300-succinylated proteins were found in the cytoplasm, which were primarily enriched in the carbohydrate metabolism process. Interestingly, deleting led to a notable decrease in Ksucc levels on several glycolytic enzymes, especially Phosphoglycerate Kinase 1 (). Mutation of the succinylated site of notably hindered cell glycolysis and lactic acid excretion. Metabolomics in vivo indicated that p300-caused metabolic reprogramming was mainly attributed to the altered carbohydrate metabolism. In addition, 89.35% of LUAD patients exhibited cytoplasmic localization of p300, with higher levels in tumor tissues than adjacent normal tissues. High levels of p300 correlated with advanced tumor stages and poor prognosis of LUAD patients. Briefly, we disclose the activity of p300 to catalyze succinylation, which contributes to cell glucose metabolic reprogramming and malignant progression of lung cancer.
Topics: Humans; Adenocarcinoma of Lung; Glucose; Lung Neoplasms; Metabolic Reprogramming; E1A-Associated p300 Protein
PubMed: 38256128
DOI: 10.3390/ijms25021057 -
Free Radical Biology & Medicine Jun 2024Neuronal energy metabolism dysregulation is involved in various pathologies of Ischemia-reperfusion (I/R), yet the role of RGMA in neuronal metabolic reprogramming has...
Neuronal energy metabolism dysregulation is involved in various pathologies of Ischemia-reperfusion (I/R), yet the role of RGMA in neuronal metabolic reprogramming has not been reported. In this study, we found that RGMA expression significantly increased after I/R, and compared to control mice, mice with MCAO/R showed an increase in glycolytic metabolic products and the expression of glycolytic pathway proteins. Furthermore, RGMA levels are closely related to neuronal energy metabolism. We discovered that knockdown of RGMA can shift neuronal energy metabolism towards oxidative phosphorylation and the pentose phosphate pathway, thereby protecting mice from ischemic reperfusion injury. Mechanistically, knockdown of RGMA can downregulate PGK1 expression, reducing the increase in glycolytic flux following ischemia reperfusion. Moreover, we found that knockdown of RGMA can reduce the interaction between USP10 and PGK1, thus affecting the ubiquitination degradation of PGK1. In summary, our data suggest that RGMA may regulate neuronal energy metabolism by inhibiting the USP10-mediated deubiquitination of PGK1, thus protecting it from I/R injury. This study provides new ideas for clarifying the intrinsic mechanism of neuronal damage after I/R.
Topics: Animals; Humans; Male; Mice; Disease Models, Animal; Energy Metabolism; Gene Knockdown Techniques; Glycolysis; Ischemic Stroke; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Oxidative Phosphorylation; Pentose Phosphate Pathway; Phosphoglycerate Kinase; Reperfusion Injury; Ubiquitin Thiolesterase; Ubiquitination
PubMed: 38556067
DOI: 10.1016/j.freeradbiomed.2024.03.020