Protein Phosphatase 2A (PP2A) mutations in brain function, development, and neurologic disease.

By removing Ser/Thr-specific phosphorylations in a multitude of protein substrates in diverse tissues, Protein Phosphatase type 2A (PP2A) enzymes play essential regulatory roles in cellular signalling and physiology, including in brain function and development. Here, we review current knowledge on PP2A gene mutations causally involved in neurodevelopmental disorders and intellectual disability, focusing on PPP2CA, PPP2R1A and PPP2R5D. We provide insights into the impact of these mutations on PP2A structure, substrate specificity and potential function in neurobiology and brain development.

The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction.

PURPOSE: Neurodevelopmental disorders (NDD) caused by protein phosphatase 2A (PP2A) dysfunction have mainly been associated with de novo variants in PPP2R5D and PPP2CA, and more rarely in PPP2R1A. Here, we aimed to better understand the latter by characterizing 30 individuals with de novo and often recurrent variants in this PP2A scaffolding Aα subunit. METHODS: Most cases were identified through routine clinical diagnostics. Variants were biochemically characterized for phosphatase activity and interaction with other PP2A subunits. RESULTS: We describe 30 individuals with 16 different variants in PPP2R1A, 21 of whom had variants not previously reported. The severity of developmental delay ranged from mild learning problems to severe intellectual disability (ID) with or without epilepsy. Common features were language delay, hypotonia, and hypermobile joints. Macrocephaly was only seen in individuals without B55α subunit-binding deficit, and these patients had less severe ID and no seizures. Biochemically more disruptive variants with impaired B55α but increased striatin binding were associated with profound ID, epilepsy, corpus callosum hypoplasia, and sometimes microcephaly. CONCLUSION: We significantly expand the phenotypic spectrum of PPP2R1A-related NDD, revealing a broader clinical presentation of the patients and that the functional consequences of the variants are more diverse than previously reported.

The PPP2R1A cancer hotspot mutant p.R183W increases clofarabine resistance in uterine serous carcinoma cells by a gain-of-function mechanism.

PURPOSE: Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. METHODS AND RESULTS: USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. CONCLUSIONS: While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC.

Clinical and molecular characteristics of a novel rare de novo variant in PPP2CA in a patient with a developmental disorder, autism, and epilepsy.

PP2A-related (neuro) developmental disorders are a family of genetic diseases caused by a heterozygous alteration in one of several genes encoding a subunit of type 2A protein phosphatases. Reported affected genes, so far, are PPP2R5D, encoding the PP2A regulatory B56δ subunit; PPP2R1A, encoding the scaffolding Aα subunit; and PPP2CA, encoding the catalytic Cα subunit-in that order of frequency. Patients with a pathogenic de novo mutation in one of these genes, in part, present with overlapping features, such as generalized hypotonia, intellectual and developmental delay, facial dysmorphologies, seizures, and autistic features, and, in part, with opposite features, e.g., smaller versus larger head sizes or normal versus absent corpus callosum. Molecular variant characterization has been consistent so far with loss-of-function or dominant-negative disease mechanisms for all three affected genes. Here, we present a case report of another PPP2CA-affected individual with a novel de novo missense variant, resulting in a one-amino acid substitution in the Cα subunit: p.Cys196Arg. Biochemical characterization of the variant revealed its pathogenicity, as it appeared severely catalytically impaired, showed mildly affected A subunit binding, and moderately decreased binding to B/B55, B"/PR72, and all B56 subunits, except B56γ1. Carboxy-terminal methylation appeared unaffected, as was binding to B"'/STRN3-all being consistent with a partial loss of function. Clinically, the girl presented with mild-to-moderate developmental delay, a full-scale IQ of 83, mild dysmorphic facial features, tonic-clonic seizures, and autistic behaviors. Brain MRI appeared normal. We conclude that this individual falls within the milder end of the clinical and molecular spectrum of previously reported PPP2CA cases.

Peroxisome-Derived Hydrogen Peroxide Modulates the Sulfenylation Profiles of Key Redox Signaling Proteins in Flp-In T-REx 293 Cells.

The involvement of peroxisomes in cellular hydrogen peroxide (H2O2) metabolism has been a central theme since their first biochemical characterization by Christian de Duve in 1965. While the role of H2O2 substantially changed from an exclusively toxic molecule to a signaling messenger, the regulatory role of peroxisomes in these signaling events is still largely underappreciated. This is mainly because the number of known protein targets of peroxisome-derived H2O2 is rather limited and testing of specific targets is predominantly based on knowledge previously gathered in related fields of research. To gain a broader and more systematic insight into the role of peroxisomes in redox signaling, new approaches are urgently needed. In this study, we have combined a previously developed Flp-In T-REx 293 cell system in which peroxisomal H2O2 production can be modulated with a yeast AP-1-like-based sulfenome mining strategy to inventory protein thiol targets of peroxisome-derived H2O2 in different subcellular compartments. By using this approach, we identified more than 400 targets of peroxisome-derived H2O2 in peroxisomes, the cytosol, and mitochondria. We also observed that the sulfenylation kinetics profiles of key targets belonging to different protein families (e.g., peroxiredoxins, annexins, and tubulins) can vary considerably. In addition, we obtained compelling but indirect evidence that peroxisome-derived H2O2 may oxidize at least some of its targets (e.g., transcription factors) through a redox relay mechanism. In conclusion, given that sulfenic acids function as key intermediates in H2O2 signaling, the findings presented in this study provide valuable insight into how peroxisomes may be integrated into the cellular H2O2 signaling network.