Structural Integrity of Nucleolin Is Required to Suppress TDP-43-Mediated Cytotoxicity in Yeast and Human Cell Models.

The Transactivating response (TAR) element DNA-binding of 43 kDa (TDP-43) is mainly implicated in the regulation of gene expression, playing multiple roles in RNA metabolism. Pathologically, it is implicated in amyotrophic lateral sclerosis and in a class of neurodegenerative diseases broadly going under the name of frontotemporal lobar degeneration (FTLD). A common hallmark of most forms of such diseases is the presence of TDP-43 insoluble inclusions in the cell cytosol. The molecular mechanisms of TDP-43-related cell toxicity are still unclear, and the contribution to cell damage from either loss of normal TDP-43 function or acquired toxic properties of protein aggregates is yet to be established. Here, we investigate the effects on cell viability of FTLD-related TDP-43 mutations in both yeast and mammalian cell models. Moreover, we focus on nucleolin (NCL) gene, recently identified as a genetic suppressor of TDP-43 toxicity, through a thorough structure/function characterization aimed at understanding the role of NCL domains in rescuing TDP-43-induced cytotoxicity. Using functional and biochemical assays, our data demonstrate that the N-terminus of NCL is necessary, but not sufficient, to exert its antagonizing effects on TDP-43, and further support the relevance of the DNA/RNA binding central region of the protein. Concurrently, data suggest the importance of the NCL nuclear localization for TDP-43 trafficking, possibly related to both TDP-43 physiology and toxicity.

Nucleolin Rescues TDP-43 Toxicity in Yeast and Human Cell Models.

TDP-43 is a nuclear protein involved in pivotal processes, extensively studied for its implication in neurodegenerative disorders. TDP-43 cytosolic inclusions are a common neuropathologic hallmark in amyotrophic lateral sclerosis (ALS) and related diseases, and it is now established that TDP-43 misfolding and aggregation play a key role in their etiopathology. TDP-43 neurotoxic mechanisms are not yet clarified, but the identification of proteins able to modulate TDP-43-mediated damage may be promising therapeutic targets for TDP-43 proteinopathies. Here we show by the use of refined yeast models that the nucleolar protein nucleolin (NCL) acts as a potent suppressor of TDP-43 toxicity, restoring cell viability. We provide evidence that NCL co-expression is able to alleviate TDP-43-induced damage also in human cells, further supporting its beneficial effects in a more consistent pathophysiological context. Presented data suggest that NCL could promote TDP-43 nuclear retention, reducing the formation of toxic cytosolic TDP-43 inclusions.

The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel-Lindau tumor suppressor.

Mutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1 α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein-protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). We found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already shown for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell metabolic activity and necrosis, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data show an isoform-specific interaction of pVHL with MDM2, suggesting an interplay between these two E3 ubiquitin ligases.

A novel PMCA3 mutation in an ataxic patient with hypomorphic phosphomannomutase 2 (PMM2) heterozygote mutations: Biochemical characterization of the pump defect.

The neuron-restricted isoform 3 of the plasma membrane Ca2+ ATPase plays a major role in the regulation of Ca2+ homeostasis in the brain, where the precise control of Ca2+ signaling is a necessity. Several function-affecting genetic mutations in the PMCA3 pump associated to X-linked congenital cerebellar ataxias have indeed been described. Interestingly, the presence of co-occurring mutations in additional genes suggest their synergistic action in generating the neurological phenotype as digenic modulators of the role of PMCA3 in the pathologies. Here we report a novel PMCA3 mutation (G733R substitution) in the catalytic P-domain of the pump in a patient affected by non-progressive ataxia, muscular hypotonia, dysmetria and nystagmus. Biochemical studies of the pump have revealed impaired ability to control cellular Ca2+ handling both under basal and under stimulated conditions. A combined analysis by homology modeling and molecular dynamics have revealed a role for the mutated residue in maintaining the correct 3D configuration of the local structure of the pump. Mutation analysis in the patient has revealed two additional function-impairing compound heterozygous missense mutations (R123Q and G214S substitution) in phosphomannomutase 2 (PMM2), a protein that catalyzes the isomerization of mannose 6-phosphate to mannose 1-phosphate. These mutations are known to be associated with Type Ia congenital disorder of glycosylation (PMM2-CDG), the most common group of disorders of N-glycosylation. The findings highlight the association of PMCA3 mutations to cerebellar ataxia and strengthen the possibility that PMCAs act as digenic modulators in Ca2+-linked pathologies.

Novel interactions of the von Hippel-Lindau (pVHL) tumor suppressor with the CDKN1 family of cell cycle inhibitors.

Germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor predisposes patients to develop different highly vascularized cancers. pVHL targets the hypoxia-inducible transcription factor (HIF-1α) for degradation, modulating the activation of various genes involved in hypoxia response. Hypoxia plays a relevant role in regulating cell cycle progression, inducing growth arrest in cells exposed to prolonged oxygen deprivation. However, the exact molecular details driving this transition are far from understood. Here, we present novel interactions between pVHL and the cyclin-dependent kinase inhibitor family CDKN1 (p21, p27 and p57). Bioinformatics analysis, yeast two-hybrid screening and co-immunoprecipitation assays were used to predict, dissect and validate the interactions. We found that the CDKN1 proteins share a conserved region mimicking the HIF-1α motif responsible for pVHL binding. Intriguingly, a p27 site-specific mutation associated to cancer is shown to modulate this novel interaction. Our findings suggest a new connection between the pathways regulating hypoxia and cell cycle progression.

Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive metabolic disorder usually presenting in the neonatal period with intermittent episodes of hyperammonemia, psychomotor delay, and progressive encephalopathy. Adult cases usually evolve into frank spastic paraparesis. The syndrome is caused by mutations in SLC25A15/ORNT1 encoding the mitochondrial ornithine transporter; a second ornithine transporter, ORNT2 of unknown function, is also present in most placental mammals. ORNT2 is believed to originate from an ancient retro-transposition event. In yeast Saccharomyces cerevisiae the major function of the transporter (encoded by Arg11) is to shuttle ornithine from the mitochondrial matrix to the cytosol. Its inactivation abolishes growth in the absence of arginine.In this work, we used functional complementation in S. cerevisiae to characterize the function of human ORNT2 and to test the pathogenicity of ORNT1 mutations found in HHH patients. Notably, we found that human ORNT1 but not ORNT2 complements the deletion of the yeast gene, despite their high level of homology. However, we identified some key residues in ORNT2, which may recover its functional competence when replaced with the corresponding residues of ORNT1, suggesting that roles of the two transporters are different. Moreover, we used this system to test a series of missense mutations of ORNT1 identified in patients with HHH syndrome. All mutations had a detrimental effect on the functionality of the human gene, without however clear genotype-phenotype correlations. Our data support yeast as a simple and effective model to validate missense mutations occurring in patients with HHH.