Mid-old cells are a potential target for anti-aging interventions in the elderly.

The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.

Engineered elastin-like polypeptide improves the efficiency of adipose-derived stem cell-mediated cutaneous wound healing in type II diabetes mellitus.

Impaired cutaneous wound healing is a major complication in patients with diabetes mellitus (DM), leading to increased amputation and mortality rates in affected patients. Adipose-derived stem cells (ASCs) are widely used seed cells for promoted tissue regeneration to improve wound closure under diabetic conditions. However, ASCs-based therapies remain limited due to difficulties in maintaining cell quality during transplantation. To overcome this problem, extracellular matrix mimetic biomaterials have been developed for use in biomedical engineering field, including tissue engineering and regenerative medicine. Herein, a biosynthesized arginine-glycine-aspartate amino acid residues (RGD motif, known as a cell adhesion motif)-containing elastin-like polypeptides (REPs) improved the efficacy of ASCs in enhancing wound closure and skin elasticity in diabetic wounds by promoting the expression of angiogenic growth factors. Therefore, REPs can be used as potential supplements to stem cell-based therapeutic approach to accelerate diabetic wound repair.

Enhancing Viability of Human Embryonic Stem Cells during Cryopreservation via RGD-REP-Mediated Activation of FAK/AKT/FoxO3a Signaling Pathway.

BACKGROUND: Cryopreservation is a crucial method for long-term storage and stable allocation of human pluripotent stem cells (hPSCs), which are increasingly being used in various applications. However, preserving hPSCs in cryogenic conditions is challenging due to reduced recovery rates. METHODS: To address this issue, the Arginine-Glycine-Aspartate (RGD) motif was incorporated into a recombinant elastin-like peptide (REP). Human embryonic stem cells (hESCs) were treated with REP containing RGD motif (RGD-REP) during suspension and cryopreservation, and the survival rate was analyzed. The underlying mechanisms were also investigated. RESULTS: The addition of RGD-REP to the cryopreservation solution improved cell survival and pluripotency marker expression. The improvement was confirmed to be due to the activation of the FAK-AKT cascade by RGD-REP binding to hESC surface interin protein, and consequent inhibition of FoxO3a. The inactivation of FoxO3a reduced the expression of apoptosis-related genes, such as BIM, leading to increased survival of PSCs in a suspension state. CONCLUSION: RGD-REP, as a ligand for integrin protein, improves the survival and maintenance of hPSCs during cryopreservation by activating survival signals via the RGD motif. These results have potential implications for improving the efficiency of stem cell usage in both research and therapeutic applications.

Epigenetic Regulation of the Expression of T Cell Stimulatory and Inhibitory Factors by Histone H3 Lysine Modification Enzymes and Its Prognostic Roles in Glioblastoma.

BACKGROUND: This study aimed to identify the specific T cell co-stimulatory and co-inhibitory factors that play prognostic roles in patients with glioblastoma. Additionally, the unique histone H3 modification enzymes that regulate the expression levels of these specific co-stimulatory and co-inhibitory factors were investigated. METHODS: The medical records of 84 patients newly diagnosed with glioblastoma at our institution from January 2006 to December 2020 were retrospectively reviewed. Immunohistochemical (IHC) staining for T cell co-stimulatory factors (CD27, CD28, CD137, OX40, and ICOS), T cell co-inhibitory factors (CTLA4, PD1, PD-L1, TIM3, and CD200R), and histone H3 lysine modification enzymes (MLL4, RIZ, EZH1, NSD2, KDM5c, JMJD1a, UTX, and JMJD5) was performed on archived paraffin-embedded tissues obtained by biopsy or resection. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed for specific factors, which demonstrated causal relationships, in order to validate the findings of the IHC examinations. RESULTS: The mean follow-up duration was 27.5 months (range, 4.1-43.5 months). During this period, 76 patients (90.5%) died, and the mean OS was 19.4 months (95% confidence interval, 16.3-20.9 months). Linear positive correlations were observed between the expression levels of CD28 and JMJD1a (R2 linear = 0.982) and those of CD137 and UTX (R2 linear = 1.528). Alternatively, significant negative correlations were observed between the expression levels of CTLA4 and RIZ (R2 linear = -1.746) and those of PD-L1 and EZH1 (R2 linear = -2.118); these relationships were confirmed by qRT-PCR. In the multivariate analysis, increased expression levels of CD28 (P = 0.042), and CD137 (P = 0.009), and decreased expression levels of CTLA4 (P = 0.003), PD-L1 (P = 0.020), and EZH1 (P = 0.040) were significantly associated with longer survival. CONCLUSION: These findings suggest that the expression of certain T cell co-stimulatory factors, such as CD28 and CD 137, and co-inhibitory factors, such as CTLA4 and PD-L1 are associated with prognosis of glioblastoma patients.

ANT2 Accelerates Cutaneous Wound Healing in Aged Skin by Regulating Energy Homeostasis and Inflammation.

An effective healing response is critical to healthy aging. In particular, energy homeostasis has become increasingly recognized as a factor in effective skin regeneration. ANT2 is a mediator of adenosine triphosphate import into mitochondria for energy homeostasis. Although energy homeostasis and mitochondrial integrity are critical for wound healing, the role played by ANT2 in the repair process had not been elucidated to date. In our study, we found that ANT2 expression decreased in aged skin and cellular senescence. Interestingly, overexpression of ANT2 in aged mouse skin accelerated the healing of full-thickness cutaneous wounds. In addition, upregulation of ANT2 in replicative senescent human diploid dermal fibroblasts induced their proliferation and migration, which are critical processes in wound healing. Regarding energy homeostasis, ANT2 overexpression increased the adenosine triphosphate production rate by activating glycolysis and induced mitophagy. Notably, ANT2-mediated upregulation of HSPA6 in aged human diploid dermal fibroblasts downregulated proinflammatory genes that mediate cellular senescence and mitochondrial damage. This study shows a previously uncharacterized physiological role of ANT2 in skin wound healing by regulating cell proliferation, energy homeostasis, and inflammation. Thus, our study links energy metabolism to skin homeostasis and reports, to the best of our knowledge, a previously unreported genetic factor that enhances wound healing in an aging model.

N-myc downstream regulated gene 1 (ndrg1) functions as a molecular switch for cellular adaptation to hypoxia.

Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation, which may preserve ATP in the kidney and ionocytes and contribute to energy homeostasis. Lastly, we show that sodium azide treatment, which increases lactate levels under normoxia, is sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a is essential for hypoxia adaptation and functions downstream of lactate signaling to induce NKA degradation, a process known to conserve cellular energy.

Co-inhibition of ATM and ROCK synergistically improves cell proliferation in replicative senescence by activating FOXM1 and E2F1.

The multifaceted nature of senescent cell cycle arrest necessitates the targeting of multiple factors arresting or promoting the cell cycle. We report that co-inhibition of ATM and ROCK by KU-60019 and Y-27632, respectively, synergistically increases the proliferation of human diploid fibroblasts undergoing replicative senescence through activation of the transcription factors E2F1 and FOXM1. Time-course transcriptome analysis identified FOXM1 and E2F1 as crucial factors promoting proliferation. Co-inhibition of the kinases ATM and ROCK first promotes the G2/M transition via FOXM1 activation, leading to accumulation of cells undergoing the G1/S transition via E2F1 activation. The combination of both inhibitors increased this effect more significantly than either inhibitor alone, suggesting synergism. Our results demonstrate a FOXM1- and E2F1-mediated molecular pathway enhancing cell cycle progression in cells with proliferative potential under replicative senescence conditions, and treatment with the inhibitors can be tested for senomorphic effect in vivo.

Control of topoisomerase II activity and chemotherapeutic inhibition by TCA cycle metabolites.

Topoisomerase II (topo II) is essential for disentangling newly replicated chromosomes. DNA unlinking involves the physical passage of one duplex through another and depends on the transient formation of double-stranded DNA breaks, a step exploited by frontline chemotherapeutics to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our work reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with ramifications for the clinical use of anti-topo II therapies.

Hypo-trimethylation of Histone H3 Lysine 4 and Hyper-tri/dimethylation of Histone H3 Lysine 27 as Epigenetic Markers of Poor Prognosis in Patients with Primary Central Nervous System Lymphoma.

PURPOSE: This study aimed to investigate the methylation status of major histone modification sites in primary central nervous system lymphoma (PCNSL) samples and examine their prognostic roles in patients with PCNSL. MATERIALS AND METHODS: Between 2007 and 2020, 87 patients were histopathologically diagnosed with PCNSL. We performed immunohistochemical staining of the formalin-fixed paraffin-embedded samples of PCNSL for major histone modification sites, such as H3K4, H3K9, H3K27, H3K14, and H3K36. After detection of meaningful methylation sites, we examined histone modification enzymes that induce methylation or demethylation at each site using immunohistochemical staining. The meaningful immunoreactivity was validated by western blotting using fresh tissue of PCNSL. RESULTS: More frequent recurrences were found in hypomethylation of H3K4me3 (p=0.004) and hypermethylation of H3K27me2 (p<0.001) and H3K27me3 (p=0.002). These factors were also statistically related to short PFS and overall survival in the univariate and multivariate analyses. Next, histone modification enzymes inducing the demethylation of H3K4 (lysine-specific demethylase-1/2 and Jumonji AT-rich interactive domain [JARID] 1A-D]) and methylation of H3K27 (enhancer of zeste homolog [EZH]-1/2) were immu- nohistochemically stained. Among them, the immunoreactivity of JARID1A inversely associated with the methylation status of H3K4me3 (R2=-1.431), and immunoreactivity of EZH2 was directly associated with the methylation status of H3K27me2 (R2=0.667) and H3K27me3 (R2=0.604). These results were validated by western blotting in fresh PCNSL samples. CONCLUSION: Our study suggests that hypomethylation of H3K4me3 and hypermethylation of H3K27me2 and H3K27me3 could be associated with poor outcomes in patients with PCNSL and that these relationships are modified by JARID1A and EZH2.

ATM mediated-p53 signaling pathway forms a novel axis for senescence control.

Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, our data indicated that p53 pathway functions as potential target for ATM-mediated senescence amelioration.

Global transcriptional downregulation of TREX and nuclear trafficking machinery as pan-senescence phenomena: evidence from human cells and tissues.

Nucleocytoplasmic trafficking (NCT) of macromolecules is a fundamental process in eukaryotes that requires tight controls to maintain proper cell functions. Downregulation of the classical NCT pathway in senescent cells has been reported. However, whether this is a hallmark that exists across all types of cellular senescence remains unknown, and whether the mRNA export machinery is altered during senescence has not been demonstrated. Here, we show that the global transcriptomic downregulation of both the TREX (transcription-export) machinery and classical NLS-dependent protein transport machinery is a hallmark of varying types of senescence. A gene set-based approach using 25 different studies showed that the TREX-NCT gene set displays distinct common downregulated patterns in senescent cells versus its expression in their nonsenescent counterparts regardless of the senescence type, such as replicative senescence (RS), tumor cell senescence (TCS), oncogene-induced senescence (OIS), stem cell senescence (SCS), progeria and endothelial cell senescence (ECS). Similar patterns of TREX-NCT gene downregulation were also shown in two large human tissue genomic databases, the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases. We also found that early-stage cancer tissues show consistent age-related patterns of TREX-NCT enrichment, suggesting the potential significance of TREX-NCT genes in determining cell fate in the early stage of tumorigenesis. Moreover, human cancer tissues exhibit an opposite TREX-NCT enrichment pattern with aging, indicating that deviation from age-related changes in TREX-NCT genes may provide a novel but critical clue for the age-dependent pathogenesis of cancer and increase in cancer incidence with aging.

Indoprofen prevents muscle wasting in aged mice through activation of PDK1/AKT pathway.

BACKGROUND: Muscle wasting, resulting from aging or pathological conditions, leads to reduced quality of life, increased morbidity, and increased mortality. Much research effort has been focused on the development of exercise mimetics to prevent muscle atrophy and weakness. In this study, we identified indoprofen from a screen for peroxisome proliferator-activated receptor γ coactivator α (PGC-1α) inducers and report its potential as a drug for muscle wasting. METHODS: The effects of indoprofen treatment on dexamethasone-induced atrophy in mice and in 3-phosphoinositide-dependent protein kinase-1 (PDK1)-deleted C2C12 myotubes were evaluated by immunoblotting to determine the expression levels of myosin heavy chain and anabolic-related and oxidative metabolism-related proteins. Young, old, and disuse-induced muscle atrophic mice were administered indoprofen (2 mg/kg body weight) by gavage. Body weight, muscle weight, grip strength, isometric force, and muscle histology were assessed. The expression levels of muscle mass-related and function-related proteins were analysed by immunoblotting or immunostaining. RESULTS: In young (3-month-old) and aged (22-month-old) mice, indoprofen treatment activated oxidative metabolism-related enzymes and led to increased muscle mass. Mechanistic analysis using animal models and muscle cells revealed that indoprofen treatment induced the sequential activation of AKT/p70S6 kinase (S6K) and AMP-activated protein kinase (AMPK), which in turn can augment protein synthesis and PGC-1α induction, respectively. Structural prediction analysis identified PDK1 as a target of indoprofen and, indeed, short-term treatment with indoprofen activated the PDK1/AKT/S6K pathway in muscle cells. Consistent with this finding, PDK1 inhibition abrogated indoprofen-induced AKT/S6K activation and hypertrophic response. CONCLUSIONS: Our findings demonstrate the effects of indoprofen in boosting skeletal muscle mass through the sequential activation of PDK1/AKT/S6K and AMPK/PGC-1α. Taken together, our results suggest that indoprofen represents a potential drug to prevent muscle wasting and weakness related to aging or muscle diseases.

Satellite cell-specific ablation of Cdon impairs integrin activation, FGF signalling, and muscle regeneration.

BACKGROUND: Perturbation in cell adhesion and growth factor signalling in satellite cells results in decreased muscle regenerative capacity. Cdon (also called Cdo) is a component of cell adhesion complexes implicated in myogenic differentiation, but its role in muscle regeneration remains to be determined. METHODS: We generated inducible satellite cell-specific Cdon ablation in mice by utilizing a conditional Cdon allele and Pax7 CreERT2 . To induce Cdon ablation, mice were intraperitoneally injected with tamoxifen (tmx). Using cardiotoxin-induced muscle injury, the effect of Cdon depletion on satellite cell function was examined by histochemistry, immunostaining, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Isolated myofibers or myoblasts were utilized to determine stem cell function and senescence. To determine pathways related to Cdon deletion, injured muscles were subjected to RNA sequencing analysis. RESULTS: Satellite cell-specific Cdon ablation causes impaired muscle regeneration with fibrosis, likely attributable to decreased proliferation, and senescence, of satellite cells. Cultured Cdon-depleted myofibers exhibited 32 ± 9.6% of EdU-positive satellite cells compared with 58 ± 4.4% satellite cells in control myofibers (P < 0.05). About 32.5 ± 3.7% Cdon-ablated myoblasts were positive for senescence-associated ß-galactosidase (SA-ß-gal) while only 3.6 ± 0.5% of control satellite cells were positive (P < 0.001). Transcriptome analysis of muscles at post-injury Day 4 revealed alterations in genes related to mitogen-activated protein kinase signalling (P < 8.29 e-5 ) and extracellular matrix (P < 2.65 e-24 ). Consistent with this, Cdon-depleted tibialis anterior muscles had reduced phosphorylated extracellular signal-regulated kinase (p-ERK) protein levels and expression of ERK targets, such as Fos (0.23-fold) and Egr1 (0.31-fold), relative to mock-treated control muscles (P < 0.001). Cdon-depleted myoblasts exhibited impaired ERK activation in response to basic fibroblast growth factor. Cdon ablation resulted in decreased and/or mislocalized integrin ß1 activation in satellite cells (weak or mislocalized integrin1 in tmx = 38.7 ± 1.9%, mock = 21.5 ± 6%, P < 0.05), previously linked with reduced fibroblast growth factor (FGF) responsiveness in aged satellite cells. In mechanistic studies, Cdon interacted with and regulated cell surface localization of FGFR1 and FGFR4, likely contributing to FGF responsiveness of satellite cells. Satellite cells from a progeria model, Zmpste24-/- myofibers, showed decreased Cdon levels (Cdon-positive cells in Zmpste24-/- = 63.3 ± 11%, wild type = 90 ± 7.7%, P < 0.05) and integrin ß1 activation (weak or mislocalized integrin ß1 in Zmpste24-/- = 64 ± 6.9%, wild type = 17.4 ± 5.9%, P < 0.01). CONCLUSIONS: Cdon deficiency in satellite cells causes impaired proliferation of satellite cells and muscle regeneration via aberrant integrin and FGFR signalling.

Systematic identification of an integrative network module during senescence from time-series gene expression.

BACKGROUND: Cellular senescence irreversibly arrests growth of human diploid cells. In addition, recent studies have indicated that senescence is a multi-step evolving process related to important complex biological processes. Most studies analyzed only the genes and their functions representing each senescence phase without considering gene-level interactions and continuously perturbed genes. It is necessary to reveal the genotypic mechanism inferred by affected genes and their interaction underlying the senescence process. RESULTS: We suggested a novel computational approach to identify an integrative network which profiles an underlying genotypic signature from time-series gene expression data. The relatively perturbed genes were selected for each time point based on the proposed scoring measure denominated as perturbation scores. Then, the selected genes were integrated with protein-protein interactions to construct time point specific network. From these constructed networks, the conserved edges across time point were extracted for the common network and statistical test was performed to demonstrate that the network could explain the phenotypic alteration. As a result, it was confirmed that the difference of average perturbation scores of common networks at both two time points could explain the phenotypic alteration. We also performed functional enrichment on the common network and identified high association with phenotypic alteration. Remarkably, we observed that the identified cell cycle specific common network played an important role in replicative senescence as a key regulator. CONCLUSIONS: Heretofore, the network analysis from time series gene expression data has been focused on what topological structure was changed over time point. Conversely, we focused on the conserved structure but its context was changed in course of time and showed it was available to explain the phenotypic changes. We expect that the proposed method will help to elucidate the biological mechanism unrevealed by the existing approaches.

Chemical screening identifies ATM as a target for alleviating senescence.

Senescence, defined as irreversible cell-cycle arrest, is the main driving force of aging and age-related diseases. Here, we performed high-throughput screening to identify compounds that alleviate senescence and identified the ataxia telangiectasia mutated (ATM) inhibitor KU-60019 as an effective agent. To elucidate the mechanism underlying ATM's role in senescence, we performed a yeast two-hybrid screen and found that ATM interacted with the vacuolar ATPase V1 subunits ATP6V1E1 and ATP6V1G1. Specifically, ATM decreased E-G dimerization through direct phosphorylation of ATP6V1G1. Attenuation of ATM activity restored the dimerization, thus consequently facilitating assembly of the V1 and V0 domains with concomitant reacidification of the lysosome. In turn, this reacidification induced the functional recovery of the lysosome/autophagy system and was coupled with mitochondrial functional recovery and metabolic reprogramming. Together, our data reveal a new mechanism through which senescence is controlled by the lysosomal-mitochondrial axis, whose function is modulated by the fine-tuning of ATM activity.

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ.

DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1-526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to that of the wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to that of the wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Km for dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site) but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to that of the wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment, i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions but not opposite G and 8-oxoG, in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations.

Succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-Phosphate (SAICAR) Activates Pyruvate Kinase Isoform M2 (PKM2) in Its Dimeric Form.

Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy about how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2(G415R), a variant derived from a cancer patient, which we show by size-exclusion chromatography and small-angle X-ray scattering to be a dimer that cannot form a tetramer in solution. Although PKM2(G415R) binds to fructose 1,6-bisphosphate (FBP), unlike the wild type this PKM2 variant shows no activation by FBP. In contrast, PKM2(G415R) is activated by succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-phosphate (SAICAR), an endogenous metabolite that we previously showed correlates with an increased level of cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression.

Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.

Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η- cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.

SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells.

Abnormal metabolism and sustained proliferation are hallmarks of cancer. Pyruvate kinase M2 (PKM2) is a metabolic enzyme that plays important roles in both processes. Recently, PKM2 was shown to have protein kinase activity phosphorylating histone H3 and promoting cancer cell proliferation. However, the mechanism and extent of this protein kinase in cancer cells remain unclear. Here, we report that binding of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR), a metabolite abundant in proliferating cells, induces PKM2's protein kinase activity in vitro and in cells. Protein microarray experiments revealed that more than 100 human proteins, mostly protein kinases, are phosphorylated by PKM2-SAICAR. In particular, PKM2-SAICAR phosphorylates and activates Erk1/2, which in turn sensitizes PKM2 for SAICAR binding through phosphorylation. Additionally, PKM2-SAICAR was necessary to induce sustained Erk1/2 activation and mitogen-induced cell proliferation. Thus, the ligand-induced protein kinase activity from PKM2 is a mechanism that directly couples cell proliferation with intracellular metabolic status.