Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein.

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.

Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines.

In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCNPOS and FLCNNEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCNNEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.

mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

Lysine methylation by the mitochondrial methyltransferase FAM173B optimizes the function of mitochondrial ATP synthase.

Lysine methylation is an important post-translational modification that is also present on mitochondrial proteins, but the mitochondrial lysine-specific methyltransferases (KMTs) responsible for modification are in most cases unknown. Here, we set out to determine the function of human family with sequence similarity 173 member B (FAM173B), a mitochondrial methyltransferase (MTase) reported to promote chronic pain. Using bioinformatics analyses and biochemical assays, we found that FAM173B contains an atypical, noncleavable mitochondrial targeting sequence responsible for its localization to mitochondria. Interestingly, CRISPR/Cas9-mediated KO of FAM173B in mammalian cells abrogated trimethylation of Lys-43 in ATP synthase c-subunit (ATPSc), a modification previously reported as ubiquitous among metazoans. ATPSc methylation was restored by complementing the KO cells with enzymatically active human FAM173B or with a putative FAM173B orthologue from the nematode Caenorhabditis elegans Interestingly, lack of Lys-43 methylation caused aberrant incorporation of ATPSc into the ATP synthase complex and resulted in decreased ATP-generating ability of the complex, as well as decreased mitochondrial respiration. In summary, we have identified FAM173B as the long-sought KMT responsible for methylation of ATPSc, a key protein in cellular ATP production, and have demonstrated functional significance of ATPSc methylation. We suggest renaming FAM173B to ATPSc-KMT (gene name ATPSCKMT).

MDH1 deficiency is a metabolic disorder of the malate-aspartate shuttle associated with early onset severe encephalopathy.

The reversible oxidation of L-malate to oxaloacetate is catalyzed by NAD(H)-dependent malate dehydrogenase (MDH). MDH plays essential roles in the malate-aspartate shuttle and the tricarboxylic acid cycle. These metabolic processes are important in mitochondrial NADH supply for oxidative phosphorylation. Recently, bi-allelic mutations in mitochondrial MDH2 were identified in patients with global developmental delay, epilepsy and lactic acidosis. We now report two patients from an extended consanguineous family with a deleterious variant in the cytosolic isoenzyme of MDH (MDH1). The homozygous missense variant in the NAD+-binding domain of MDH1 led to severely diminished MDH protein expression. The patients presented with global developmental delay, epilepsy and progressive microcephaly. Both patients had normal concentrations of plasma amino acids, acylcarnitines, lactate, and urine organic acids. To identify the metabolic consequences of MDH1 deficiency, untargeted metabolomics was performed on dried blood spots (DBS) from the patients and in MDH1 knockout HEK293 cells that were generated by Crispr/Cas9. Increased levels of glutamate and glycerol-3-phosphate were found in DBS of both patients. In MDH1 KO HEK293 cells, increased levels of glycerol-3-phosphate were also observed, as well as increased levels of aspartate and decreased levels of fumarate. The consistent finding of increased concentrations of glycerol-3-phosphate may represent a compensatory mechanism to enhance cytosolic oxidation of NADH by the glycerol-P-shuttle. In conclusion, MDH1 deficiency is a new metabolic defect in the malate-aspartate shuttle characterized by a severe neurodevelopmental phenotype with elevated concentrations of glycerol-3-phosphate as a potential biomarker.

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion.

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Rheb and Rags come together at the lysosome to activate mTORC1.

mTORC1 (mammalian target of rampamycin complex 1) is a highly conserved protein complex regulating cell growth and metabolism via its kinase mTOR (mammalian target of rapamycin). The activity of mTOR is under the control of various GTPases, of which Rheb and the Rags play a central role. The presence of amino acids is a strict requirement for mTORC1 activity. The heterodimeric Rag GTPases localize mTORC1 to lysosomes by their amino-acid-dependent interaction with the lysosomal Ragulator complex. Rheb is also thought to reside on lysosomes to activate mTORC1. Rheb is responsive to growth factors, but, in conjunction with PLD1 (phospholipase D1), is also an integral part of the machinery that stimulates mTORC1 in response to amino acids. In the present article, we provide a brief overview of novel mechanisms by which amino acids affect the function of Rags. On the basis of existing literature, we postulate that Rheb is activated at the Golgi from where it will travel to lysosomes. Maturation of endosomes into lysosomes may be required to assure a continuous supply of GTP-bound Rheb for mTORC1 activation, which may help to drive the maturation process.

Metabolic Alterations in NADSYN1-Deficient Cells.

NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway.

The malate-aspartate shuttle is important for de novo serine biosynthesis.

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD+. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-13C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD+/NADH ratio), consistent with the NAD+ dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD+-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders.

The long-term effect of mTOR inhibition on lipid and glucose metabolism in tuberous sclerosis complex: data from the Dutch TSC registry.

BACKGROUND: MTOR inhibition is an effective treatment for many manifestations of tuberous sclerosis complex. Because mTOR inhibition is a disease modifying therapy, lifelong use will most likely be necessary. This study addresses the long-term effects of mTOR inhibitors on lipid and glucose metabolism and aims to provide better insight in the incidence and time course of these metabolic adverse effects in treated TSC patients. METHODS: All patients who gave informed consent for the nationwide TSC Registry and were ever treated with mTOR inhibitors (sirolimus and/or everolimus) were included. Lipid profiles, HbA1c and medication were analysed in all patients before and during mTOR inhibitor treatment. RESULTS: We included 141 patients, the median age was 36 years, median use of mTOR inhibitors 5.1 years (aimed serum levels 3.0-5.0 µg/l). Total cholesterol, LDL- and HDL-cholesterol levels at baseline were similar to healthy reference data. After start of mTOR inhibition therapy, total cholesterol, LDL-cholesterol and triglycerides increased significantly and were higher compared to healthy reference population. Mean total cholesterol levels increased by 1.0 mmol/L after 3-6 months of mTOR inhibition therapy but did not increase further during follow-up. In this study, 2.5% (3/118) of patients developed diabetes (defined as an HbA1c ≥ 48 mmol/mol) during a median follow-up of 5 years. CONCLUSIONS: Hypercholesterolemia is a frequent side effect of mTOR inhibition in TSC patients, and predominantly occurs within the first year of treatment. Although hyperglycemia is a frequent side effect in other indications for mTOR inhibition, incidence of diabetes mellitus in TSC patients was only 2.5%. This may reflect the difference of mTOR inhibition in patients with normal mTOR complex pathway function versus patients with overactive mTOR complex signaling due to a genetic defect (TSC patients).

Low nociceptor GRK2 prolongs prostaglandin E2 hyperalgesia via biased cAMP signaling to Epac/Rap1, protein kinase Cepsilon, and MEK/ERK.

Hyperexcitability of peripheral nociceptive pathways is often associated with inflammation and is an important mechanism underlying inflammatory pain. Here we describe a completely novel mechanism via which nociceptor G-protein-coupled receptor kinase 2 (GRK2) contributes to regulation of inflammatory hyperalgesia. We show that nociceptor GRK2 is downregulated during inflammation. In addition, we show for the first time that prostaglandin E2 (PGE2)-induced hyperalgesia is prolonged from <6 h in wild-type (WT) mice to 3 d in mice with low GRK2 in Nav1.8+ nociceptors (SNS-GRK2+/- mice). This prolongation of PGE2 hyperalgesia in SNS-GRK2+/- mice does not depend on changes in the sensitivity of the prostaglandin receptors because prolonged hyperalgesia also developed in response to 8-Br-cAMP. PGE2 or cAMP-induced hyperalgesia in WT mice is PKA dependent. However, PKA activity is not required for hyperalgesia in SNS-GRK2+/- mice. SNS-GRK2+/- mice developed prolonged hyperalgesia in response to the Exchange proteins directly activated by cAMP (Epac) activator 8-pCPT-2'-O-Me-cAMP (8-pCPT). Coimmunoprecipitation experiments showed that GRK2 binds to Epac1. In vitro, GRK2 deficiency increased 8-pCPT-induced activation of the downstream effector of Epac, Rap1, and extracellular signal-regulated kinase (ERK). In vivo, inhibition of MEK1 or PKCε prevented prolonged PGE2, 8-Br-cAMP, and 8-pCPT hyperalgesia in SNS-GRK2+/- mice. In conclusion, we discovered GRK2 as a novel Epac1-interacting protein. A reduction in the cellular level of GRK2 enhances activation of the Epac-Rap1 pathway. In vivo, low nociceptor GRK2 leads to prolonged inflammatory hyperalgesia via biased cAMP signaling from PKA to Epac-Rap1, ERK/PKCε pathways.

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans.

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell-cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the alpha-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration.

Evolution of the TOR pathway.

The TOR kinase is a major regulator of growth in eukaryotes. Many components of the TOR pathway are implicated in cancer and metabolic diseases in humans. Analysis of the evolution of TOR and its pathway may provide fundamental insight into the evolution of growth regulation in eukaryotes and provide a practical framework on which experimental evidence can be compared between species. Here we performed phylogenetic analyses on the components of the TOR pathway and determined their point of invention. We find that the two TOR complexes and a large part of the TOR pathway originated before the Last Eukaryotic Common Ancestor and form a core to which new inputs have been added during animal evolution. In addition, we provide insight into how duplications and sub-functionalization of the S6K, RSK, SGK and PKB kinases shaped the complexity of the TOR pathway. In yeast we identify novel AGC kinases that are orthologous to the S6 kinase. These results demonstrate how a vital signaling pathway can be both highly conserved and flexible in eukaryotes.

Upregulation of the pathogenic transcription factor SPI1/PU.1 in tuberous sclerosis complex and focal cortical dysplasia by oxidative stress.

Tuberous sclerosis complex (TSC) is a congenital disorder characterized by cortical malformations and concomitant epilepsy caused by loss-of-function mutations in the mTOR suppressors TSC1 or TSC2. While the underlying molecular changes caused by mTOR activation in TSC have previously been investigated, the drivers of these transcriptional change have not been fully elucidated. A better understanding of the perturbed transcriptional regulation could lead to the identification of novel pathways for therapeutic intervention not only in TSC, but other genetic epilepsies in which mTOR activation plays a key role, such as focal cortical dysplasia 2b (FCD). Here, we analyzed RNA sequencing data from cortical tubers and a tsc2-/- zebrafish. We identified differential expression of the transcription factors (TFs) SPI1/PU.1, IRF8, GBX2, and IKZF1 of which SPI1/PU.1 and IRF8 targets were enriched among the differentially expressed genes. Furthermore, for SPI1/PU.1 these findings were conserved in TSC zebrafish model. Next, we confirmed overexpression of SPI1/PU.1 on the RNA and protein level in a separate cohort of surgically resected TSC tubers and FCD tissue, in fetal TSC tissue, and a Tsc1GFAP-/- mouse model of TSC. Subsequently, we validated the expression of SPI1/PU.1 in dysmorphic cells with mTOR activation in TSC tubers. In fetal TSC, we detected SPI1/PU.1 expression prenatally and elevated RNA Spi1 expression in Tsc1GFAP-/- mice before the development of seizures. Finally, in vitro, we identified that in astrocytes and neurons SPI1 transcription was driven by H2 O2 -induced oxidative stress, independent of mTOR. We identified SPI1/PU.1 as a novel TF involved in the pro-inflammatory gene expression of malformed cells in TSC and FCD 2b. This transcriptional program is activated in response to oxidative stress and already present prenatally. Importantly, SPI1/PU.1 protein appears to be strictly limited to malformed cells, as we did not find SPI1/PU.1 protein expression in mice nor in our in vitro models.

Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.

Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanism by which FLCN prevents kidney cancer remains unknown. Here, we show that disrupting FLCN in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.

Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.

Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662* ; VPS41c.1423-2A>G and VPS41R662* ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662* abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.

Cell-cell junction formation: the role of Rap1 and Rap1 guanine nucleotide exchange factors.

Rap proteins are Ras-like small GTP-binding proteins that amongst others are involved in the control of cell-cell and cell-matrix adhesion. Several Rap guanine nucleotide exchange factors (RapGEFs) function to activate Rap. These multi-domain proteins, which include C3G, Epacs, PDZ-GEFs, RapGRPs and DOCK4, are regulated by various different stimuli and may function at different levels in junction formation. Downstream of Rap, a number of effector proteins have been implicated in junctional control, most notably the adaptor proteins AF6 and KRIT/CCM1. In this review, we will highlight the latest findings on the Rap signaling network in the control of epithelial and endothelial cell-cell junctions.

Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity.

BACKGROUND: Retrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks. RESULTS: Here we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12th and 59th amino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio in vivo, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen. CONCLUSIONS: Together, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitt's lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS) warrants further investigation into its role in disease development.