MITOCHONDRIAL VITIATION CONGRUENTLY APTLY WITH AUTISM SPECTRUM DISORDER.

Mitochondrial dysfunction in autism leads to impair the mitochondria's ability to synthesis adenosine triphosphate (ATP) by impairment citric acid cycle as well as increase anaerobic glycolysis. Aim - measuring and evaluating the levels of mitochondrial markers; including glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), malate dehydrogenase, and pyruvate kinase) in the autistic group and knowing the possibility of using these markers to diagnose children with autism spectrum disorder. A case-control study was done in the Al-Zahraa Teaching Hospital (Kut City, Iraq) on 100 Iraqi children (male and female), between (April 2023 and January 2024). Their ages ranged between 3 and 9 years. Among them were 50 patients enrolled as autistic group and 50 healthy enrolled as control group. Blood samples were collected and bioassays for GOT, GPT, pyruvate kinase, and malate dehydrogenase were measured by ELISA technique. The autistic group showed that the urine GOT, urine GPT, serum malate, and serum pyruvate levels in the ASD group was significantly higher (P<0.001) than the control group. The ROC analysis showed that urine GOT, urine GOT, serum malate and serum pyruvate had an accuracy level of (81%,71%,77%, and 80 %) and the area under the curve (AUC) was > 0.7 (0.8),0.7, 0.7(0.76), and 0.7(0.8) thus urine GOT, urine GPT, serum, malate, and serum pyruvate are a valid diagnostic marker. There was a significant difference in the mean urine and serum concentrations of mitochondrial markers (GOT, GPT, malate dehydrogenase, and pyruvate kinase) between autistic children and the control group due to mitochondrial dysfunction.

Expression and Significance of PKM1 in Acute Myeloid Leukaemia.

OBJECTIVE: To investigate the expression level of pyruvate kinase M1 (PKM1) in patients with acute myeloid leukaemia (AML) as well as its clinical significance. STUDY DESIGN: A case-control study. Place and Duration of the Study: Department of Haematology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China, from January 2013 to 2023. METHODOLOGY: The expression levels of PKM1 and pyruvate kinase m2 (PKM2) in the bone marrow of 65 AML patients (excluding M3) and 31 healthy volunteers were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), a method that measures fluorescence in real-time. The associations between PKM1, PKM2 expressions, clinical parameters, and the survival and prognosis of AML patients were analysed. RESULTS: AML patients showed higher PKM1 expression compared to controls. The area under the curve (AUC) of the receiver operating characteristics (ROC) was 0.65 (p = 0.017). PKM1 expression was correlated with peripheral blood leukocyte count (r = -0.276, p = 0.026), CCAAT enhancer-binding protein alpha CEBPA mutation (r = -0.306, p = 0.014), and chemotherapy-induced response (r = -0.292, p = 0.018). Patients with high PKM1 expression had a lower remission rate (p = 0.019) and long-term survival rate (p = 0.034) than those with low PKM1 expression. Patients with AML showed a rise in PKM2 levels; however, the variation was not statistically significant (p >0.05). CONCLUSION: PKM1 expression is upregulated in AML and patients with high PKM1 expression have a lower survival rate. KEY WORDS: PKM1, Acute myeloid leukaemia, Clinical prognosis.

The role of NAD-dependent deacetylase sirtuin-2 in liver metabolic stress through regulating pyruvate kinase M2 ubiquitination.

NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2.

PKM2 aggregation drives metabolism reprograming during aging process.

While protein aggregation's association with aging and age-related diseases is well-established, the specific proteins involved and whether dissolving them could alleviate aging remain unclear. Our research addresses this gap by uncovering the role of PKM2 aggregates in aging. We find that PKM2 forms aggregates in senescent cells and organs from aged mice, impairing its enzymatic activity and glycolytic flux, thereby driving cells into senescence. Through a rigorous two-step small molecule library screening, we identify two compounds, K35 and its analog K27, capable of dissolving PKM2 aggregates and alleviating senescence. Further experiments show that treatment with K35 and K27 not only alleviate aging-associated signatures but also extend the lifespan of naturally and prematurely aged mice. These findings provide compelling evidence for the involvement of PKM2 aggregates in inducing cellular senescence and aging phenotypes, and suggest that targeting these aggregates could be a promising strategy for anti-aging drug discovery.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Neurotoxic effects of triclosan in adolescent mice: Pyruvate kinase M2 dimer regulated Signal transducer and activator of transcription 3 phosphorylation mediated microglia activation and neuroinflammation.

Triclosan (TCS), a commonly used antibacterial agent, is associated with various harmful effects on mammalian neurodevelopment, particularly when exposed prenatally. This study investigated the impact of long-term exposure to TCS on the prefrontal cortex development in adolescent mice. We evaluated the motor ability, motor coordination, and anxiety behavior of mice using open field tests (OFT) and elevated cross maze tests (EPM). An increase in movement distance, number of passes through the central area, and open arm retention time was observed in mice treated with TCS. Hematoxylin eosin staining and Nissl staining also showed significant adverse reactions in the brain tissue of TCS-exposed group. TCS induced microglia activation and increased inflammatory factors expression in the prefrontal cortex. TCS also increased the expression of pyruvate kinase M2 (PKM2), thereby elevating the levels of PKM2 dimer, which entered the nucleus. Treatment with TEPP46 (PKM2 dimer nuclear translocation inhibitor) blocked the expression of inflammatory factors induced by TCS. TCS induced the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro, upregulating the levels of inflammatory cytokines. The results also demonstrated the binding of PKM2 to STAT3, which promoted STAT3 phosphorylation at the Tyr705 site, thereby regulating the expression of inflammatory factors. These findings highlight the role of PKM2-regulated STAT3 phosphorylation in TCS-induced behavioral disorders in adolescents and propose a reliable treatment target for TCS.

Compound 3K attenuates isoproterenol-induced cardiac hypertrophy by inhibiting pyruvate kinase M2 (PKM2) pathway.

AIM: Chronic sympathetic stimulation has been identified as a primary factor in the pathogenesis of cardiac hypertrophy (CH). However, there is no appropriate treatment available for the management of CH. Recently, it has been revealed that pyruvate kinase M2 (PKM2) plays a significant role in cardiac remodeling, fibrosis, and hypertrophy. However, the therapeutic potential of selective PKM2 inhibitor has not yet been explored in cardiac hypertrophy. Thus, in the current study, we have studied the cardioprotective potential of Compound 3K, a selective PKM2 inhibitor in isoproterenol-induced CH model. METHODS: To induce cardiac hypertrophy, male Wistar rats were subcutaneously administered isoproterenol (ISO, 5 mg/kg/day) for 14 days. Compound 3K at dosages of 2 and 4 mg/kg orally was administered to ISO-treated rats for 14 days to explore its effects on various parameters like ECG, ventricular functions, hypertrophic markers, histology, inflammation, and protein expression were performed. RESULTS: Fourteen days administration of ISO resulted in the induction of CH, which was evidenced by alterations in ECG, ventricular dysfunctions, increase in hypertrophy markers, and fibrosis. The immunoblotting of hypertrophy heart revealed the significant rise in PKM2 and reduction in PKM1 protein expression. Treatment with Compound 3K led to downregulation of PKM2 and upregulation of PKM1 protein expression. Compound 3K showed cardioprotective effects by improving ECG, cardiac functions, hypertrophy markers, inflammation, and fibrosis. Further, it also reduced cardiac expression of PKM2-associated splicing protein, HIF-1α, and caspase-3. CONCLUSION: Our findings suggest that Compound 3K has a potential cardioprotective effect via PKM2 inhibition in isoproterenol-induced CH.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

Rheostatic contributions to protein stability can obscure a position's functional role.

Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.

Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation.

The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2TG) mice, PHB2S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.

Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C-Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. METHODS AND RESULTS: Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37) and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) in vitro and in vivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. CONCLUSIONS: This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Apigenin intervenes in liver fibrosis by regulating PKM2-HIF-1α mediated oxidative stress.

Apigenin (API) is a natural flavonoid compound with antioxidant, anti fibrotic, anti-inflammatory and other effects, but there is limited research on the effect of API on liver fibrosis. This study aims to explore the effect and potential mechanism of API on liver fibrosis induced by CCl4 in mice. The results indicate that API reduces oxidative stress levels, inhibits hepatic stellate cell (HSC) activation, and exerts anti liver fibrosis effects by regulating the PKM2-HIF-1α pathway. We observed that API alleviated liver tissue pathological damage and collagen deposition in CCl4 induced mouse liver fibrosis model, promoting the recovery of liver function in mice with liver fibrosis. In addition, the API inhibits the transition of Pyruvate kinase isozyme type M2 (PKM2) from dimer to tetramer formation by regulating the EGFR-MEK1/2-ERK1/2 pathway, thereby preventing dimer from entering the nucleus and blocking PKM2-HIF-1α access. This change leads to a decrease in malondialdehyde (MDA) and Catalase (CAT) levels and an increase in glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) levels, as well as total antioxidant capacity (T-AOC) in the liver of liver fibrosis mice. At the same time, API downregulated the expression of α-smooth muscle actin (α-SMA), Vimentin and Desmin in the liver tissue of mice with liver fibrosis, inhibited the activation of HSC, and reduced collagen deposition. These results indicate that API can inhibit HSC activation and alleviate CCl4 induced liver fibrosis by inhibiting the PKM2-HIF-1α pathway and reducing oxidative stress, laying an important foundation for the development and clinical application of API as a novel drug for treating liver fibrosis.

Pyruvate kinase deficiency in 29 Turkish patients with two novel intronic variants.

Pyruvate kinase (PK) is a key enzyme of anaerobic glycolysis. The genetic heterogeneity of PK deficiency (PKD) is high, and over 400 unique variants have been identified. Twenty-nine patients who had been diagnosed as PKD genetically in seven distinct paediatric haematology departments were evaluated. Fifteen of 23 patients (65.2%) had low PK levels. The PK:hexokinase ratio had 100% sensitivity for PKD diagnosis, superior to PK enzyme assay. Two novel intronic variants (c.695-1G>A and c.694+43C>T) have been described. PKD should be suspected in patients with chronic non-spherocytic haemolytic anaemia, even if enzyme levels are falsely normal. Total PKLR gene sequencing is necessary for the characterization of patients with PKD and for genetic counselling.

Oridonin attenuates liver ischemia-reperfusion injury by suppressing PKM2/NLRP3-mediated macrophage pyroptosis.

BACKGROUND: The NOD-like receptor protein 3 (NLRP3) mediated pyroptosis of macrophages is closely associated with liver ischemia reperfusion injury (IRI). As a covalent inhibitor of NLRP3, Oridonin (Ori), has strong anti-inflammasome effect, but its effect and mechanisms for liver IRI are still unknown. METHODS: Mice and liver macrophages were treated with Ori, respectively. Co-IP and LC-MS/MS analysis of the interaction between PKM2 and NLRP3 in macrophages. Liver damage was detected using H&E staining. Pyroptosis was detected by WB, TEM, and ELISA. RESULTS: Ori ameliorated liver macrophage pyroptosis and liver IRI. Mechanistically, Ori inhibited the interaction between pyruvate kinase M2 isoform (PKM2) and NLRP3 in hypoxia/reoxygenation（H/R）-induced macrophages, while the inhibition of PKM2/NLRP3 reduced liver macrophage pyroptosis and liver IRI. CONCLUSION: Ori exerted protective effects on liver IRI via suppressing PKM2/NLRP3-mediated liver macrophage pyroptosis, which might become a potential therapeutic target in the clinic.

TIPE1 limits virus replication by disrupting PKM2/ HIF-1α/ glycolysis feedback loop.

OBJECTIVE: Virus infection is a major threat to human health and remains a significant cause of death to date. Macrophages are important innate immune cells that exhibit indispensable roles in controlling virus replication. It was recently reported that metabolic adaption determines the functional state of macrophages. Thus, to further unravel the crucial factors involving in metabolic adaption of macrophages might provide the potential candidates for optimizing their anti-viral capabilities. METHODS: RT-PCR, Western blotting, virus plaque assay and HE were used to evaluate the viral load in virus-infected Tipe1M-KO and Tipe1f/f mice or cultured macrophages. RNA sequencing were performed with Tipe1M-KOor Tipe1f/f BMDMs upon virus infection. Extracellular acidification rate (ECAR) was applied for analyzing glycolysis rate in virus-infected BMDMs. Co-immunoprecipitation (Co-IP) assay and LC-MS/MS were used to determine the potential interacting proteins of TIPE1. RESULTS: TIPE1 level was significantly reduced in BMDMs infected with either RNA viruses or DNA virus. Deficiency of Tipe1 in macrophages increased viral load and aggravated tissue damage. Mechanistically, TIPE1 suppressed the glycolytic capacity of macrophages through interacting with PKM2 and promoting its ubiquitination degradation, which in turn decreased HIF1α transcription and viral replication in macrophages. CONCLUSIONS: TIPE1 functions as a novel regulator for metabolic reprogramming and virus infection in macrophages.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

The Regulatory Network of hnRNPs Underlying Regulating PKM Alternative Splicing in Tumor Progression.

One of the hallmarks of cancer is metabolic reprogramming in tumor cells, and aerobic glycolysis is the primary mechanism by which glucose is quickly transformed into lactate. As one of the primary rate-limiting enzymes, pyruvate kinase (PK) M is engaged in the last phase of aerobic glycolysis. Alternative splicing is a crucial mechanism for protein diversity, and it promotes PKM precursor mRNA splicing to produce PKM2 dominance, resulting in low PKM1 expression. Specific splicing isoforms are produced in various tissues or illness situations, and the post-translational modifications are linked to numerous disorders, including cancers. hnRNPs are one of the main components of the splicing factor families. However, there have been no comprehensive studies on hnRNPs regulating PKM alternative splicing. Therefore, this review focuses on the regulatory network of hnRNPs on PKM pre-mRNA alternative splicing in tumors and clinical drug research. We elucidate the role of alternative splicing in tumor progression, prognosis, and the potential mechanism of abnormal RNA splicing. We also summarize the drug targets retarding tumorous splicing events, which may be critical to improving the specificity and effectiveness of current therapeutic interventions.

Clinically meaningful improvements in patient-reported outcomes in mitapivat-treated patients with pyruvate kinase deficiency.

Clinically meaningful benefits in the signs, symptoms, and impacts of #PKDeficiency as assessed by disease-specific patient-reported outcome measures were observed in mitapivat-treated adult patients in two phase 3 clinical trials.