Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function.

NF2 (moesin-ezrin-radixin-like [MERLIN] tumor suppressor) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here, we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in NF2 with clustered mutations which affected conformation-dependent protein interactions. NF2 variants in the F2-F3 subdomain and the α3H helix region substantially modulated NF2 conformation and homomerization. Mutations in the F2-F3 subdomain altered proliferation in three cell lines and matched patterns of disease mutations in NF2 related-schwannomatosis. This study highlights the power of systematic mutational interaction perturbation analysis to identify missense variants impacting NF2 conformation and provides insight into NF2 tumor suppressor function.

Interaction modulation through arrays of clustered methyl-arginine protein modifications.

Systematic analysis of human arginine methylation identifies two distinct signaling modes; either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions.

Augmentation of phosphate-induced osteo-/chondrogenic transformation of vascular smooth muscle cells by homoarginine.

AIMS: Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification. METHODS AND RESULTS: Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine. CONCLUSION: Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide.

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50 values of ∼2.4·10(-5) and ∼7.9·10(-5) m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ∼1.5·10(-5) m at pH 6.0 and increased to ∼8.3·10(-5) m at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with an IC50 of ∼1.9·10(-5) m in the presence of Arg and BH4 but exhibited much higher IC50 values (∼1.0-6.1·10(-4) m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC50 ∼ 1.3-2.0·10(-5) m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation.

Aerobic nitric oxide-induced thiol nitrosation in the presence and absence of magnesium cations.

Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg(2+). In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9 ± 0.4 and 0.55 ± 0.06 M(-1)s(-1) in the presence and absence of Mg(2+). The maximum yield of GSNO was close to 100% in the presence of Mg(2+) but only about half as high in its absence. From this observation we conclude that, in the absence of Mg(2+), nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg(2+), demonstrating O2(-) formation. The reaction in the presence of Mg(2+) seems to involve formation of a Mg(2+)•glutathione (GSH) complex. SOD did not affect Mg(2+)-stimulated nitrosation, suggesting that no O2(-) is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg(2+)-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.

Interaction between neuronal nitric-oxide synthase and tetrahydrobiopterin revisited: studies on the nature and mechanism of tight pterin binding.

Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10(-7)-10(-6) M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10(-7) M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 µM BH4 (2 min with 1 µM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.