TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase.

The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.

mTOR Regulates Phase Separation of PGL Granules to Modulate Their Autophagic Degradation.

The assembly of phase-separated structures is thought to play an important role in development and disease, but little is known about the regulation and function of phase separation under physiological conditions. We showed that during C. elegans embryogenesis, PGL granules assemble via liquid-liquid phase separation (LLPS), and their size and biophysical properties determine their susceptibility to autophagic degradation. The receptor SEPA-1 promotes LLPS of PGL-1/-3, while the scaffold protein EPG-2 controls the size of PGL-1/-3 compartments and converts them into less dynamic gel-like structures. Under heat-stress conditions, mTORC1-mediated phosphorylation of PGL-1/-3 is elevated and PGL-1/-3 undergo accelerated phase separation, forming PGL granules that are resistant to autophagic degradation. Significantly, accumulation of PGL granules is an adaptive response to maintain embryonic viability during heat stress. We revealed that mTORC1-mediated LLPS of PGL-1/-3 acts as a switch-like stress sensor, coupling phase separation to autophagic degradation and adaptation to stress during development.

Coenzyme A precursors flow from mother to zygote and from microbiome to host.

Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.

Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation.

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.

Direction of leukocyte polarization and migration by the phosphoinositide-transfer protein TIPE2.

The polarization of leukocytes toward chemoattractants is essential for the directed migration (chemotaxis) of leukocytes. How leukocytes acquire polarity after encountering chemical gradients is not well understood. We found here that leukocyte polarity was generated by TIPE2 (TNFAIP8L2), a transfer protein for phosphoinositide second messengers. TIPE2 functioned as a local enhancer of phosphoinositide-dependent signaling and cytoskeleton remodeling, which promoted leading-edge formation. Conversely, TIPE2 acted as an inhibitor of the GTPase Rac, which promoted trailing-edge polarization. Consequently, TIPE2-deficient leukocytes were defective in polarization and chemotaxis, and TIPE2-deficient mice were resistant to leukocyte-mediated neural inflammation. Thus, the leukocyte polarizer is a dual-role phosphoinositide-transfer protein and represents a potential therapeutic target for the treatment of inflammatory diseases.

Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

Control of the innate immune response by the mevalonate pathway.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1ß that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS.

Mutations in the genes encoding pyrin and mevalonate kinase (MVK) cause distinct interleukin-1ß (IL-1ß)-mediated autoinflammatory diseases: familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS). Pyrin forms an inflammasome when mutant or in response to bacterial modification of the GTPase RhoA. We found that RhoA activated the serine-threonine kinases PKN1 and PKN2 that bind and phosphorylate pyrin. Phosphorylated pyrin bound to 14-3-3 proteins, regulatory proteins that in turn blocked the pyrin inflammasome. The binding of 14-3-3 and PKN proteins to FMF-associated mutant pyrin was substantially decreased, and the constitutive IL-1ß release from peripheral blood mononuclear cells of patients with FMF or HIDS was attenuated by activation of PKN1 and PKN2. Defects in prenylation, seen in HIDS, led to RhoA inactivation and consequent pyrin inflammasome activation. These data suggest a previously unsuspected fundamental molecular connection between two seemingly distinct autoinflammatory disorders.

Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors.

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

Diet-induced developmental acceleration independent of TOR and insulin in C. elegans.

Dietary composition has major effects on physiology. Here, we show that developmental rate, reproduction, and lifespan are altered in C. elegans fed Comamonas DA1877 relative to those fed a standard E. coli OP50 diet. We identify a set of genes that change in expression in response to this diet and use the promoter of one of these (acdh-1) as a dietary sensor. Remarkably, the effects on transcription and development occur even when Comamonas DA1877 is diluted with another diet, suggesting that Comamonas DA1877 generates a signal that is sensed by the nematode. Surprisingly, the developmental effect is independent from TOR and insulin signaling. Rather, Comamonas DA1877 affects cyclic gene expression during molting, likely through the nuclear hormone receptor NHR-23. Altogether, our findings indicate that different bacteria elicit various responses via distinct mechanisms, which has implications for diseases such as obesity and the interactions between the human microbiome and intestinal cells.

Integration of metabolic and gene regulatory networks modulates the C. elegans dietary response.

Expression profiles are tailored according to dietary input. However, the networks that control dietary responses remain largely uncharacterized. Here, we combine forward and reverse genetic screens to delineate a network of 184 genes that affect the C. elegans dietary response to Comamonas DA1877 bacteria. We find that perturbation of a mitochondrial network composed of enzymes involved in amino acid metabolism and the TCA cycle affects the dietary response. In humans, mutations in the corresponding genes cause inborn diseases of amino acid metabolism, most of which are treated by dietary intervention. We identify several transcription factors (TFs) that mediate the changes in gene expression upon metabolic network perturbations. Altogether, our findings unveil a transcriptional response system that is poised to sense dietary cues and metabolic imbalances, illustrating extensive communication between metabolic networks in the mitochondria and gene regulatory networks in the nucleus.

Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

A phosphoinositide signalling pathway mediates rapid lysosomal repair.

Lysosomal dysfunction has been increasingly linked to disease and normal ageing1,2. Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors3. Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function.

PI3K drives the de novo synthesis of coenzyme A from vitamin B5.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

Genome-wide Screening Identifies SFMBT1 as an Oncogenic Driver in Cancer with VHL Loss.

von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.

Ectopic assembly of an auxin efflux control machinery shifts developmental trajectories.

Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.

Tocopherol and phylloquinone biosynthesis in chloroplasts requires the phytol kinase VITAMIN E PATHWAY GENE5 (VTE5) and the farnesol kinase (FOLK).

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK.

PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes.

How cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2. As PI(4,5)P2 decreased, actin was depleted from the membrane, allowing secretion. Forced localization of PIP5Kß across the synapse prevented actin depletion, blocking both centrosome docking and secretion. Thus, PIP5Ks act as molecular sensors of TCR activation, controlling actin recruitment across the synapse, ensuring exquisite co-ordination between TCR signaling and CTL secretion.

The human microbiome encodes resistance to the antidiabetic drug acarbose.

The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search strategy, we discovered that bacterial members of the human gut and oral microbiome encode enzymes that selectively phosphorylate a clinically used antidiabetic drug, acarbose1,2, resulting in its inactivation. Acarbose is an inhibitor of both human and bacterial α-glucosidases3, limiting the ability of the target organism to metabolize complex carbohydrates. Using biochemical assays, X-ray crystallography and metagenomic analyses, we show that microbiome-derived acarbose kinases are specific for acarbose, provide their harbouring organism with a protective advantage against the activity of acarbose, and are widespread in the microbiomes of western and non-western human populations. These results provide an example of widespread microbiome resistance to a non-antibiotic drug, and suggest that acarbose resistance has disseminated in the human microbiome as a defensive strategy against a potential endogenous producer of a closely related molecule.