The molecular elements that govern cellular transformation and tumorigenic competence remain poorly understood. Metabolic reprogramming has emerged as a hallmark of malignant transformation. Recently ...in Cell Metabolism, Zhang et al. showed that an increase of cellular antioxidant capacity and nucleotide availability is sufficient to induce oncogenic transformation and tumorigenesis.
NAD
kinases (NADKs) are metabolite kinases that phosphorylate NAD
molecules to make NADP
, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH ...production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.
Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. ...Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.
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•The small intestine has high de novo synthesis, while kidney shows high purine salvage•The de novo synthesis and salvage pathway contribute similarly to tumor purine pools•The purine salvage pathway plays a crucial role in promoting tumor growth•Dietary supplementation of nucleotides accelerates tumor growth
Comprehensive isotope tracing analyses reveal the contribution of de novo synthesis and salvage pathways in supplying purine nucleotides across major tissues and tumors.
Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, ...proliferation is suppressed in electron transport chain (ETC)–dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial β-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.
Editor’s summary When liver damage occurs, a key condition for recovery is ensuring the proliferation of healthy tissue without propagating sick or damaged cells. Wang et al . have identified a mechanism by which this happens through selective pressure on the mitochondria. In some pathogenic conditions, such as cirrhosis, the diseased areas contain mutations in the mitochondrial genome that lead to dysfunction in the electron transport chain required for cellular respiration. Although such dysfunction is not directly lethal, the authors have found that it puts the affected cells at a competitive disadvantage and reduces their metabolic flexibility. In addition, biliary epithelial cells can pitch in and transdifferentiate into hepatocytes when healthy liver cells are in short supply. —Yevgeniya Nusinovich
INTRODUCTION Mitochondrial electron transport chain (ETC) dysfunction is commonly observed in acquired human diseases, including in the setting of metabolic-associated liver diseases. During liver regeneration, proliferating hepatocytes compete and allow cells with increased fitness to more readily contribute to the composition of the regenerated organ. Although recent studies indicate that mitochondrial ETC function can affect stem cell behavior, it is unknown whether the ETC influences hepatocyte proliferation after liver injury and thereby contributes to the organ’s recovered condition and function. RATIONALE We used metabolite profiling of mitochondria and isotope tracing techniques in mice to investigate the metabolic response in hepatocytes under homeostatic and regenerative conditions. We then used a set of genetic mouse models targeting the mitochondrial ETC to dissect the contribution of each individual ETC complex (I to V) to liver regeneration. With this approach we aimed to examine the relative fitness of wild-type (WT) and ETC-dysfunctional hepatocytes during regeneration and identify mechanisms by which mitochondrial health is regulated in proliferating hepatocytes. RESULTS We found that mouse hepatocytes required a functional ETC to proliferate and compete with WT hepatocytes during liver regeneration. In the absence of an ETC, murine livers rapidly accumulated fatty acid species, resulting in steatosis. We additionally observed that transdifferentiation of cholangiocytes into hepatocytes was stimulated during regeneration of ETC-mutant livers. Metabolic tracing studies revealed that WT livers rely on mobilization and oxidation of peripheral fat stores to maintain acetyl-CoA levels during proliferation. In ETC-mutant livers, fatty acid oxidation was inhibited resulting in fat accumulation and decreased production of acetyl-CoA. Notably, mitochondrial complex I was not required for hepatocyte proliferation, suggesting that complex I is not the major electron donor to the ETC in regenerating hepatocytes. As fat accumulates in the setting of ETC dysfunction, the generation of acetyl-CoA from nonfatty acid sources (such as pyruvate or acetate) was suppressed as a result of induced expression of PDK4 (a negative regulator of pyruvate oxidation) and decreased expression of ACSS2 (the enzyme responsible for conversion of acetate to acetyl-CoA). This metabolic inflexibility (the inability to switch to an alternative nutrient for generation of acetyl-CoA) forces a reliance on fatty acid oxidation and thereby selects for proliferating hepatocytes with a functional ETC. To test this model we inhibited or deleted PDK4 to re-enable pyruvate oxidation to acetyl-CoA. In the absence of PDK4 activity, ETC-dysfunctional hepatocytes were able to proliferate during liver regeneration. CONCLUSION Our results support a model whereby the network topology regulating nutrient utilization in the liver encodes a metabolic inflexibility that promotes mitochondrial health during tissue regeneration. Specifically, the accumulation of fatty acids in the setting of ETC dysfunction inhibits the generation of acetyl-CoA from alternative substrates. We identify PDK4 expression downstream of fat accumulation as a key regulatory event that governs metabolic inflexibility in proliferating hepatocytes. Although metabolic flexibility has been largely proposed as beneficial to an organism’s survival and function, our model indicates that metabolic inflexibility can be used by the murine liver to promote the overall health of a population of proliferating cells. Suppressed acetyl-CoA production promotes mitochondrial health during liver regeneration. During liver injury and regeneration, fatty acids from adipose tissues transit to the liver to fuel mitochondrial beta oxidation in WT hepatocytes, which outcompete ETC-mutant hepatocytes. In the absence of a functional ETC, fatty acid buildup reduces acetyl-CoA through induction of PDK4. PDK4 inhibition restores flexibility for acetyl-CoA generation, allowing ETC-mutant hepatocytes to proliferate. Figure created with BioRender.com
Nicotinamide adenine dinucleotide phosphate (NADP
) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH ...exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP
, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.
The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these ...interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate 'lynchpins', which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the 'lynchpin' site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology. Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py.
Here, we describe a phosphorylation-based reverse myristoyl switch for mammalian ZNRF2, and show that this E3 ubiquitin ligase and its sister protein ZNRF1 regulate the Na(+)/K(+) pump ...(Na(+)/K(+)ATPase). N-myristoylation localizes ZNRF1 and ZNRF2 to intracellular membranes and enhances their activity. However, when ZNRF2 is phosphorylated in response to agonists including insulin and growth factors, it binds to 14-3-3 and is released into the cytosol. On membranes, ZNRF1 and ZNRF2 interact with the Na(+)/K(+)ATPase α1 subunit via their UBZ domains, while their RING domains interact with E2 proteins, predominantly Ubc13 that, together with Uev1a, mediates formation of Lys63-ubiquitin linkages. ZNRF1 and ZNRF2 can ubiquitylate the cytoplasmic loop encompassing the nucleotide-binding and phosphorylation regions of the Na(+)/K(+)ATPase α1 subunit. Ouabain, a Na(+)/K(+)ATPase inhibitor and therapeutic cardiac glycoside, decreases ZNRF1 protein levels, whereas knockdown of ZNRF2 inhibits the ouabain-induced decrease of cell surface and total Na(+)/K(+)ATPase α1 levels. Thus, ZNRF1 and ZNRF2 are new players in regulation of the ubiquitous Na(+)/K(+)ATPase that is tuned to changing demands in many physiological contexts.
Insulin receptor substrate 1 (IRS1) and IRS2 are well-characterized adapter proteins that relay signals from receptor tyrosine kinases to downstream components of signalling pathways. In contrast, ...the function of IRS4 is not well understood. IRS4 overexpression has been associated with acute lymphoblastic leukaemia and subungual exostosis, while point mutations of IRS4 have been found in melanomas. Here, we show that while IRS4 expression is low in most cancer cell lines, IRS4 mRNA and protein levels are markedly elevated in certain cells including the NCI-H720, DMS114, HEK293T and HEK293AAV lines. Surprisingly, IRS4 expression was also strongly induced when HEK293 cells were infected with retroviral particles and selected under puromycin, making IRS4 expression a potential off-target effect of retroviral expression vectors. Cells with high expression of IRS4 displayed high phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, as well as elevated Akt and p70 S6 kinase activities, even in the absence of growth factors. PI 3-kinase (PI3K) signalling in these cells depends on IRS4, even though these cells also express IRS1/2. Knockdown of IRS4 also inhibited cell proliferation in cells with high levels of IRS4. Together, these findings suggest IRS4 as a potential therapeutic target for cancers with high expression of this protein.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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