Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase

This is the final version. Available on open access from Portland Press via the DOI in this recordData Availability: Structure factors and co-ordinates have been deposited in the PDB with accession code 5NKP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [50] partner repository with the dataset identifier PXD031606 and 10.6019/PXD031606.The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing four amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3

nicotinamide counteracts alcohol induced impairment of hepatic protein metabolism in humans

We have recently shown that a large amount of wine (750 mL, 7 0 g of alcohol) markedly impairs postprandial hepatic protein metabolism in healthy subjects. This is probably due to the shift in the intracellular redox state (increased NADH/NAD + ) induced by ethanol oxidation. If this hypothesis is true, the administration of nicotinamide (NAD + precursor) should provide NAD + in excess and thus correct the NADH/NAD + abnormalities and prevent the ethanol hepatotoxicity. Whole-body protein metabolism and the fractional secretory rates of hepatic (albumin, fibrinogen) and extra-hepatic (immunoglobulin G, lgG) plasma proteins were measured in the basal postabsorptive and in the absorptive states in 15 healthy subjects, that had been assigned to three groups matched for age and body mass index. During the absorptive state (intragastric meal), the three groups received water (control), 750 mL of wine, or 750 mL of wine + 1.25 g of nicotinamide, respectively. The redox state was estimated by determining the plasma lactate/pyruvate ratio. Compared with the basal state, wine alone increased the lactate/pyruvate ratio twofold and depressed the fractional secretory rates of albumin and fibrinogen (P < 0.01 vs. control and nicotinamide); nicotinamide reduced the effects of wine on the lactate/pyruvate ratio (P < 0.02 vs. wine alone) and prevented the reduction of albumin and fibrinogen secretory rates (P 0.05 vs. control). These results indicate that nicotinamide counteracts the acute hepatotoxic effects of ethanol by ameliorating the redox state

The functional significance of the sodium-potassium pump in motor control and movement disorders

The sodium-potassium pump (NKA) is a ubiquitously expressed membrane protein. Mammalian neurons express two isoforms of the NKA’s catalytic α-subunit, the ubiquitous α1, and the neuron-specific α3. Mutations in the α3-NKA encoding protein ATP1A3 result in a spectrum of neurological disorders with pronounced motor deficits, including Rapid-Onset Dystonia Parkinsonism (RDP). The NKA has also been shown to facilitate activity-dependent changes in spinal motor networks via an ultra-slow afterhyperpolarisation (usAHP) which is thought to be an α3-NKA mediated current. First, we provide a comprehensive characterisation of a novel mouse model of RDP harbouring the T613M mutation of ATP1A3 most commonly found in patients. We show that T613M animals are hyperactive and hyperambulatory. We show involvement of spinal motor circuit pathology in this behaviour via a complete lack of usAHPs in T613M-affected motor neurons, and a reduced capacity for isolated spinal cords to regulate rhythmic motor output in response to large increases of intracellular sodium. We show that this deficiency is likely caused by a reduced capacity for T613M-affected α3-NKA to extrude sodium and therefore maintain sodium homeostasis. Next, we characterised post-discharge activity more generally in motor neurons from wildtype mice. We show that usAHPs are more commonly observed in the second postnatal week which may be due to an upregulation of α3-NKA protein expression in motor neurons alongside the development of weight-bearing locomotion. While the heterogenous distribution of usAHPs amongst populations of neurons was previously thought to be due to mosaicism of α3-NKA expression, we show that all lumbar motor neurons express α3-NKA. We present an alternative to this hypothesis, showing that usAHPs can be masked by competing conductances. Finally, we show that the post-discharge activity of a neuron can be modulated by extrinsic and intrinsic modulators to enhance or unmask certain post-discharge activity subtypes

Practical Considerations for Managing Pregnancy in Patients With Multiple Sclerosis: Dispelling the Myths.

PURPOSE OF REVIEW: Lack of consistent data and guidance have led to variations between clinicians in the management of pregnancy in women with multiple sclerosis (MS). Pregnant and/or lactating women are often excluded from clinical trials conducted in MS, and thus, the labeling for most disease-modifying therapies (DMTs) excludes use during pregnancy. This has led to heterogeneity in interpretation and labeling regarding the safety of DMTs during pregnancy and lactation and the required preconception washout periods. This review identifies key themes where there is conflicting information surrounding family planning and pregnancy in MS, focusing on the most common discussion points between physicians and patients during preconception planning, pregnancy, postpartum, and lactation. The goal was to inform the patient-physician conversation and provide best practice recommendations based on expert clinical expertise and experience. RECENT FINDINGS: We outline the latest evidence-based data for DMT use during pregnancy and lactation, the effect of MS on fertility and fertility treatments, the risk of adverse pregnancy and delivery outcomes, the risk of postpartum relapse, and immunization and clinical imaging safety during pregnancy and breastfeeding. SUMMARY: Management of family planning and pregnancy in patients with MS requires the most current information. Health care providers should discuss family planning early and frequently with patients with MS, and partners where practicable. Because management of pregnant people with MS will often require a risk/benefit analysis of their needs, shared decision-making in family planning discussions is emphasized. Additional data are needed for specific and underrepresented populations with MS (e.g., single parents or those from the LGBTQ+ community) and those at risk of racial and socioeconomic disparities in care. Pregnancy registries and the design and conduct of clinical trials focused on pregnant and lactating patients should provide additional data to guide the ongoing management of patients with MS