Differential Effects of Post-translational Modifications on the Membrane Interaction of Huntingtin Protein.

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple post-translational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the effect of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17-membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of the Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insights into the complex relationship between Nt17 structure, PTMs, and membrane binding.

Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.

Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington's disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.

PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.

Phosphatases of regenerating liver (PRLs) are markers of cancer and promote tumor growth. They have been implicated in a variety of biochemical pathways but the physiologically relevant target of phosphatase activity has eluded 20 years of investigation. Here, we show that PRL3 catalytic activity is not required in a mouse model of metastasis. PRL3 binds and inhibits CNNM4, a membrane protein associated with magnesium transport. Analysis of PRL3 mutants specifically defective in either CNNM-binding or phosphatase activity demonstrate that CNNM binding is necessary and sufficient to promote tumor metastasis. As PRLs do have phosphatase activity, they are in fact pseudo-pseudophosphatases. Phosphatase activity leads to formation of phosphocysteine, which blocks CNNM binding and may play a regulatory role. We show levels of PRL cysteine phosphorylation vary in response to culture conditions and in different tissues. Examination of related protein phosphatases shows the stability of phosphocysteine is a unique and evolutionarily conserved property of PRLs. The demonstration that PRL3 functions as a pseudophosphatase has important ramifications for the design of PRL inhibitors for cancer.

Mg2+-ATP Sensing in CNNM, a Putative Magnesium Transporter.

The family of cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg2+ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg2+ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg2+-ATP-analog bound and ligand free. The structures reveal open and closed conformations with functionally important contacts not observed in structures of the individual domains. We also identified a second Mg2+-binding region in the CBS-pair domain and a different dimerization interface for the CNBH domain. Analytical ultracentrifugation and isothermal titration calorimetry experiments revealed a tight correlation between Mg2+-ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg2+ efflux activity. The results suggest Mg2+ efflux is regulated by conformational changes associated with Mg2+-ATP binding to CNNM CBS-pair domains.

Mechanism of thienopyridone and iminothienopyridinedione inhibition of protein phosphatases.

Thienopyridone (TP) has been proposed as a selective inhibitor of phosphatases of regenerating liver (PRL or PTP4A). PRLs are dual specificity phosphatases that promote cancer progression and are attractive anticancer targets. TP and iminothienopyridinedione (ITP), a more potent derivative, were shown to be effective inhibitors but the mechanism of inhibition was not established. Here, we perform NMR experiments and in vitro phosphatase assays to show that TP and ITP inhibit protein phosphatases non-specifically through oxidation of the phosphatase catalytic cysteine. We demonstrate that TP and ITP are redox active compounds, inhibiting PRL-3 and multiple other PTPs through oxidation. They also catalyze the oxidation of thioredoxin-1 as well as small molecules, like TCEP, DTT, and glutathione. The reported selectivity of TP and ITP is likely due to the higher susceptibility of PRLs to oxidation. Thus, while TP and ITP effectively inhibit PRLs, their use for studying the cellular function of PRLs is problematic due to the likelihood of off-target effects.