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Experimental Cell Research Apr 2024Serine metabolic reprogramming is known to be associated with oncogenesis and tumor development. The key metabolic enzyme PSAT1 has been identified as a potential...
Serine metabolic reprogramming is known to be associated with oncogenesis and tumor development. The key metabolic enzyme PSAT1 has been identified as a potential prognostic marker for various cancers, but its role in ccRCC remains unkown. In this study, we investigated expression of PSAT1 in ccRCC using the TCGA database and clinical specimens. Our results showed that PSAT1 exhibited lower expression in tumor tissue compared to adjacent normal tissue, but its expression level increased with advancing stages and grades of ccRCC. Patients with elevated expression level of PSAT1 exhibited an unfavorable prognosis. Functional experiments have substantiated that the depletion of PSAT1 shows an effective activity in inhibiting the proliferation, migration and invasion of ccRCC cells, concurrently promoting apoptosis. RNA sequencing analysis has revealed that the attenuation of PSAT1 can diminish tumor resistance to therapeutic drugs. Furthermore, the xenograft model has indicated that the inhibition of PSAT1 can obviously impact the tumorigenic potential of ccRCC and mitigate lung metastasis. Notably, pharmacological targeting PSAT1 by Aminooxyacetic Acid (AOA) or knockdown of PSAT1 increased the susceptibility of sunitinib-resistant cells. Inhibition of PSAT1 increased the sensitivity of drug-resistant tumors to sunitinib in vivo. Collectively, our investigation identifies PSAT1 as an independent prognostic biomarker for advanced ccRCC patients and as a prospective therapeutic target.
Topics: Humans; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Drug Resistance; Kidney Neoplasms; Sunitinib; Up-Regulation
PubMed: 38373588
DOI: 10.1016/j.yexcr.2024.113977 -
Cancer Research Apr 2024Serine metabolism plays a pivotal role in cancer, making it an appealing therapeutic target. Two recent studies published in Nature Metabolism and Science Translational...
Serine metabolism plays a pivotal role in cancer, making it an appealing therapeutic target. Two recent studies published in Nature Metabolism and Science Translational Medicine uncovered novel players and therapeutic opportunities within this crucial metabolic pathway. Papalazarou and colleagues employed genetic tools coupled with metabolomics and high-throughput imaging to identify and characterize membrane transporters involved in serine uptake and mitochondrial import in colorectal cancer. Notably, they showed that dual inhibition of these transporters in combination with impaired serine biosynthesis reduced tumor growth in xenograft models. In a parallel study, Zhang and colleagues identified isocitrate dehydrogenase I (IDH1) as a novel regulator of serine biosynthesis in non-small cell lung cancer. Through extensive mechanistic studies, they demonstrated that IDH1 enhances the expression of the key enzymes phosphoglycerate dehydrogenase and phosphoserine aminotransferase 1 via a noncanonical function independent of its enzymatic activity. Strikingly, pharmacologic disruption of this novel function of IDH1 not only diminished tumor growth but also enhanced the anticancer efficacy of dietary serine restriction in mouse models of lung cancer. Together, these studies advance our mechanistic understanding of how cancer cells fulfill their serine requirements and reveal innovative therapeutic avenues to deprive tumors of this vital nutrient.
Topics: Animals; Mice; Humans; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Serine; Cell Line, Tumor; Phosphoglycerate Dehydrogenase
PubMed: 38364233
DOI: 10.1158/0008-5472.CAN-24-0541 -
Journal of Visualized Experiments : JoVE Jan 2024Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear...
Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.
Topics: Humans; Amino Acid Sequence; Amino Acids; Ligands; Peptides; Proteins; Interferon Regulatory Factors; Protein Binding; Binding Sites; Amino Acid Motifs
PubMed: 38345234
DOI: 10.3791/66314 -
Biochimica Et Biophysica Acta.... Apr 2024Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by...
Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PP) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation. Atomic force microscopy (AFM) revealed that DPPC-liposomes had spherical and smooth surface. The presence of SM and Chol elicited rough and smooth surface, respectively. NPP1 insertion produced protrusions in all the liposome surface. Maximum phosphodiesterase activity emerged at 0.082 M ionic strength, whereas maximum phosphomonohydrolase activity arose at low ionic strength. Phosphoserine-Calcium Phosphate Complex (PS-CPLX) and amorphous calcium-phosphate (ACP) induced mineral propagation in DPPC- and DPPC:SM-liposomes and in DPPC:Chol-liposomes, respectively. Mineral characterization revealed the presence of bands assigned to HAp in the mineral propagated by NPP1 harbored in DPPC-liposomes without nucleators or in DPPC:Chol-liposomes with ACP nucleators. These data show that studying how the ionic and lipidic environment affects NPP1 properties is important, especially for HAp obtained under controlled conditions in vitro.
Topics: Calcium Phosphates; Ions; Liposomes; Minerals; Phosphoric Diester Hydrolases; Phosphoric Monoester Hydrolases; Sphingomyelins; Pyrophosphatases
PubMed: 38342362
DOI: 10.1016/j.bbamem.2024.184292 -
Nucleic Acids Research Apr 2024Protein-protein and protein-rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by...
An evolutionarily conserved phosphoserine-arginine salt bridge in the interface between ribosomal proteins uS4 and uS5 regulates translational accuracy in Saccharomyces cerevisiae.
Protein-protein and protein-rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selection involves a major conformational change-domain closure-that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This has been thought a constitutive function of the ribosome ensuring consistent accuracy. Recently, the Saccharomyces cerevisiae Ctk1 cyclin-dependent kinase was demonstrated to ensure translational accuracy and Ser238 of uS5 proposed as its target. Surprisingly, Ser238 is outside the uS4-uS5 interface and no obvious mechanism has been proposed to explain its role. We show that the true target of Ctk1 regulation is another uS5 residue, Ser176, which lies in the interface opposite to Arg57 of uS4. Based on site specific mutagenesis, we propose that phospho-Ser176 forms a salt bridge with Arg57, which should increase selectivity by strengthening the interface. Genetic data show that Ctk1 regulates accuracy indirectly; the data suggest that the kinase Ypk2 directly phosphorylates Ser176. A second kinase pathway involving TORC1 and Pkc1 can inhibit this effect. The level of accuracy appears to depend on competitive action of these two pathways to regulate the level of Ser176 phosphorylation.
Topics: Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Ribosomal Proteins; Arginine; Protein Biosynthesis; Phosphoserine; Cyclin-Dependent Kinases; Phosphorylation; Evolution, Molecular; Protein Kinases
PubMed: 38340338
DOI: 10.1093/nar/gkae053 -
The Journal of Biological Chemistry Mar 2024The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon...
The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.
Topics: Humans; 14-3-3 Proteins; 3',5'-Cyclic-AMP Phosphodiesterases; Cyclic AMP-Dependent Protein Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphorylation; Phosphoserine; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit
PubMed: 38325743
DOI: 10.1016/j.jbc.2024.105725 -
Computers in Biology and Medicine Mar 2024Phosphorylation, a prevalent post-translational modification, plays a crucial role in regulating cellular activities. This process encompasses O-phosphorylation (e.g.,...
MOTIVATION
Phosphorylation, a prevalent post-translational modification, plays a crucial role in regulating cellular activities. This process encompasses O-phosphorylation (e.g., phosphoserine) and N-phosphorylation (e.g., phospho-lysine (pK), phospho-arginine (pR), and phospho-histidine (pH)). While significant research has focused on O-phosphorylation, resulting in the development of various algorithms for predicting O-phosphorylation sites with commendable performance, there has been a notable absence of models designed to predict N-phosphorylation sites. This study introduces an integrated model named DeepNphos, designed to predict N-phosphorylation sites. This model is developed based on the analysis of thousands of experimentally identified pK, pR and pH sites.
RESULTS
Observing that the Convolutional Neural Network (CNN) model, incorporating the One-Hot encoding feature, demonstrates favorable performance in comparison to other models when predicting pK, pR, and pH sites. Additionally, pK exhibits similarities to other lysine modification types, and integrating the CNN model with a deep-transfer learning (DTL) strategy based on tens of thousands of known lysine modification sites could enhance pK prediction performance. In contrast, pR exhibits little similarity to other arginine modification types, and the integration of DTL has minimal impact on pR prediction performance. Furthermore, the decision was made to refrain from incorporating the DTL strategy in predicting pH sites, given the scarcity of histidine modification sites beyond those associated with pH. The final classifiers for predicting pK, pR, and pH sites achieve AUC values of 0.856, 0.805 and 0.802 for ten-fold cross-validation, respectively. Overall, DeepNphos is the first classifier for predicting N-phosphorylation sites, accessible at https://github.com/ChangXulinmessi/DeepNPhos.
Topics: Deep Learning; Lysine; Phosphorylation; Histidine; Protein Processing, Post-Translational; Arginine
PubMed: 38295472
DOI: 10.1016/j.compbiomed.2024.108079 -
Protein Science : a Publication of the... Feb 2024Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified...
Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species.
Topics: Histidinol-Phosphatase; Amino Acid Sequence; Phylogeny; Bacterial Proteins
PubMed: 38284491
DOI: 10.1002/pro.4899 -
Biochimica Et Biophysica Acta.... Mar 2024L-Ser supply in the central nervous system of mammals mostly relies on its endogenous biosynthesis by the phosphorylated pathway (PP). Defects in any of the three...
L-Ser supply in the central nervous system of mammals mostly relies on its endogenous biosynthesis by the phosphorylated pathway (PP). Defects in any of the three enzymes operating in the pathway result in a group of neurometabolic diseases collectively known as serine deficiency disorders (SDDs). Phosphoserine phosphatase (PSP) catalyzes the last, irreversible step of the PP. Here we investigated in detail the role of physiological modulators of human PSP activity and the properties of three natural PSP variants (A35T, D32N and M52T) associated with SDDs. Our results, partially contradicting previous reports, indicate that: i. PSP is almost fully saturated with Mg under physiological conditions and fluctuations in Mg and Ca concentrations are unlikely to play a modulatory role on PSP activity; ii. Inhibition by L-Ser, albeit at play on the isolated PSP, does not exert any effect on the flux through the PP unless the enzyme activity is severely impaired by inactivating substitutions; iii. The so-far poorly investigated A35T substitution was the most detrimental, with a 50-fold reduction in catalytic efficiency, and a reduction in thermal stability (as well as an increase in the IC for L-Ser). The M52T substitution had similar, but milder effects, while the D32N variant behaved like the wild-type enzyme. iv. Predictions of the structural effects of the A35T and M52T substitutions with ColabFold suggest that they might affect the structure of the flexible helix-loop region.
Topics: Animals; Humans; Serine; Magnesium; Ions; Mammals; Dapsone; Phosphoric Monoester Hydrolases
PubMed: 38278334
DOI: 10.1016/j.bbadis.2024.167034 -
Pathogens (Basel, Switzerland) Dec 2023Strains of , an enteric parasite of poultry, vary in virulence. Here, we performed microscopy and RNA sequencing on oocysts of strains APU-1 (which exhibits more...
Strains of , an enteric parasite of poultry, vary in virulence. Here, we performed microscopy and RNA sequencing on oocysts of strains APU-1 (which exhibits more virulence) and APU-2. Although each underwent parallel development, APU-1 initially approached maturation more slowly. Each strain sporulated by hour 36; their gene expression diverged somewhat thereafter. Candidate biomarkers of viability included 58 genes contributing at least 1000 Transcripts Per Million throughout sporulation, such as cation-transporting ATPases and zinc finger domain-containing proteins. Many genes resemble constitutively expressed genes also important to Throughout sporulation, the expression of only a few genes differed between strains; these included cyclophilin A, EF-1α, and surface antigens (SAGs). Mature and immature oocysts uniquely differentially express certain genes, such as an X-Pro dipeptidyl-peptidase domain-containing protein in immature oocysts and a profilin in mature oocysts. The immature oocysts of each strain expressed more phosphoserine aminotransferase and the mature oocysts expressed more SAGs and microneme proteins. These data illuminate processes influencing sporulation in and related genera, such as , and identify biological processes which may differentiate them. Drivers of development and senescence may provide tools to assess the viability of oocysts, which would greatly benefit the poultry industry and food safety applications.
PubMed: 38276148
DOI: 10.3390/pathogens13010002