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.

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.

The neuron-restricted isoform 3 of the plasma membrane Ca ATPase plays a major role in the regulation of Ca homeostasis in the brain, where the precise control of Ca 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 Ca 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 Ca-linked pathologies.

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.