KPNB1 modulates the Machado-Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP.

Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD.

Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides.

The actin cytoskeleton powers membrane deformation during many cellular processes, such as migration, morphogenesis, and endocytosis. Membrane phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], regulate the activities of many actin-binding proteins (ABPs), including profilin, cofilin, Dia2, N-WASP, ezrin, and moesin, but the underlying molecular mechanisms have remained elusive. Moreover, because of a lack of available methodology, the dynamics of membrane interactions have not been experimentally determined for any ABP. Here, we applied a combination of biochemical assays, photobleaching/activation approaches, and atomistic molecular dynamics simulations to uncover the molecular principles by which ABPs interact with phosphoinositide-rich membranes. We show that, despite using different domains for lipid binding, these proteins associate with membranes through similar multivalent electrostatic interactions, without specific binding pockets or penetration into the lipid bilayer. Strikingly, our experiments reveal that these proteins display enormous differences in the dynamics of membrane interactions and in the ranges of phosphoinositide densities that they sense. Profilin and cofilin display transient, low-affinity interactions with phosphoinositide-rich membranes, whereas F-actin assembly factors Dia2 and N-WASP reside on phosphoinositide-rich membranes for longer periods to perform their functions. Ezrin and moesin, which link the actin cytoskeleton to the plasma membrane, bind membranes with very high affinity and slow dissociation dynamics. Unlike profilin, cofilin, Dia2, and N-WASP, they do not require high "stimulus-responsive" phosphoinositide density for membrane binding. Moreover, ezrin can limit the lateral diffusion of PI(4,5)P2 along the lipid bilayer. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely fulfill their specific functions in cytoskeletal dynamics.

Circulating metabolites and the risk of type 2 diabetes: a prospective study of 11,896 young adults from four Finnish cohorts.

AIMS/HYPOTHESIS: Metabolomics technologies have identified numerous blood biomarkers for type 2 diabetes risk in case-control studies of middle-aged and older individuals. We aimed to validate existing and identify novel metabolic biomarkers predictive of future diabetes in large cohorts of young adults. METHODS: NMR metabolomics was used to quantify 229 circulating metabolic measures in 11,896 individuals from four Finnish observational cohorts (baseline age 24-45 years). Associations between baseline metabolites and risk of developing diabetes during 8-15 years of follow-up (392 incident cases) were adjusted for sex, age, BMI and fasting glucose. Prospective metabolite associations were also tested with fasting glucose, 2 h glucose and HOMA-IR at follow-up. RESULTS: Out of 229 metabolic measures, 113 were associated with incident type 2 diabetes in meta-analysis of the four cohorts (ORs per 1 SD: 0.59-1.50; p< 0.0009). Among the strongest biomarkers of diabetes risk were branched-chain and aromatic amino acids (OR 1.31-1.33) and triacylglycerol within VLDL particles (OR 1.33-1.50), as well as linoleic n-6 fatty acid (OR 0.75) and non-esterified cholesterol in large HDL particles (OR 0.59). The metabolic biomarkers were more strongly associated with deterioration in post-load glucose and insulin resistance than with future fasting hyperglycaemia. A multi-metabolite score comprised of phenylalanine, non-esterified cholesterol in large HDL and the ratio of cholesteryl ester to total lipid in large VLDL was associated with future diabetes risk (OR 10.1 comparing individuals in upper vs lower fifth of the multi-metabolite score) in one of the cohorts (mean age 31 years). CONCLUSIONS/INTERPRETATION: Metabolic biomarkers across multiple molecular pathways are already predictive of the long-term risk of diabetes in young adults. Comprehensive metabolic profiling may help to target preventive interventions for young asymptomatic individuals at increased risk.

Molecular mechanism for inhibition of twinfilin by phosphoinositides.

Membrane phosphoinositides control organization and dynamics of the actin cytoskeleton by regulating the activities of several key actin-binding proteins. Twinfilin is an evolutionarily conserved protein that contributes to cytoskeletal dynamics by interacting with actin monomers, filaments, and the heterodimeric capping protein. Twinfilin also binds phosphoinositides, which inhibit its interactions with actin, but the underlying mechanism has remained unknown. Here, we show that the high-affinity binding site of twinfilin for phosphoinositides is located at the C-terminal tail region, whereas the two actin-depolymerizing factor (ADF)/cofilin-like ADF homology domains of twinfilin bind phosphoinositides only with low affinity. Mutagenesis and biochemical experiments combined with atomistic molecular dynamics simulations reveal that the C-terminal tail of twinfilin interacts with membranes through a multivalent electrostatic interaction with a preference toward phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), PI(4,5)P2, and PI(3,4,5)P3 This initial interaction places the actin-binding ADF homology domains of twinfilin in close proximity to the membrane and subsequently promotes their association with the membrane, thus leading to inhibition of the actin interactions. In support of this model, a twinfilin mutant lacking the C-terminal tail inhibits actin filament assembly in a phosphoinositide-insensitive manner. Our mutagenesis data also reveal that the phosphoinositide- and capping protein-binding sites overlap in the C-terminal tail of twinfilin, suggesting that phosphoinositide binding additionally inhibits the interactions of twinfilin with the heterodimeric capping protein. The results demonstrate that the conserved C-terminal tail of twinfilin is a multifunctional binding motif, which is crucial for interaction with the heterodimeric capping protein and for tethering twinfilin to phosphoinositide-rich membranes.

Are coarse-grained models apt to detect protein thermal stability? The case of OPEP force field.

We present the first investigation of the kinetic and thermodynamic stability of two homologous thermophilic and mesophilic proteins based on the coarse-grained model OPEP. The object of our investigation is a pair of G-domains of relatively large size, 200 amino acids each, with an experimental stability gap of about 40 K. The OPEP force field is able to maintain stable the fold of these relatively large proteins within the hundrend-nanosecond time scale without including external constraints. This makes possible to characterize the conformational landscape of the folded protein as well as to explore the unfolding. In agreement with all-atom simulations used as a reference, we show that the conformational landscape of the thermophilic protein is characterized by a larger number of substates with slower dynamics on the network of states and more resilient to temperature increase. Moreover, we verify the stability gap between the two proteins using replica-exchange simulations and estimate a difference between the melting temperatures of about 23 K, in fair agreement with experiment. The detailed investigation of the unfolding thermodynamics, allows to gain insight into the mechanism underlying the enhanced stability of the thermophile relating it to a smaller heat capacity of unfolding.

The OPEP protein model: from single molecules, amyloid formation, crowding and hydrodynamics to DNA/RNA systems.

The OPEP coarse-grained protein model has been applied to a wide range of applications since its first release 15 years ago. The model, which combines energetic and structural accuracy and chemical specificity, allows the study of single protein properties, DNA-RNA complexes, amyloid fibril formation and protein suspensions in a crowded environment. Here we first review the current state of the model and the most exciting applications using advanced conformational sampling methods. We then present the current limitations and a perspective on the ongoing developments.

Evidence for inefficient contraction and abnormal mitochondrial activity in sarcopenia using magnetic resonance spectroscopy.

BACKGROUND: Mitochondrial dysfunction has been implicated in sarcopenia. 31 P magnetic resonance spectroscopy (MRS) enables non-invasive measurement of adenosine triphosphate (ATP) synthesis rates to probe mitochondrial function. Here, we assessed muscle energetics in older sarcopenic and non-sarcopenic men and compared with muscle biopsy-derived markers of mitochondrial function. METHODS: Twenty Chinese men with sarcopenia (SARC, age = 73.1 ± 4.1 years) and 19 healthy aged and sex-matched controls (CON, age = 70.3 ± 4.2 years) underwent assessment of strength, physical performance, and magnetic resonance imaging. Concentrations of phosphocreatine (PCr), ATP and inorganic phosphate (Pi) as well as muscle pH were measured at rest and during an interleaved rest-exercise protocol to probe muscle mitochondrial function. Results were compared to biopsy-derived mitochondrial complex activity and expression to understand underlying metabolic perturbations. RESULTS: Despite matched muscle contractile power (strength/cross-sectional area), the ATP contractile cost was higher in SARC compared with CON (low-intensity exercise: 1.06 ± 0.59 vs. 0.57 ± 0.22, moderate: 0.93 ± 0.43 vs. 0.58 ± 0.68, high: 0.70 ± 0.57 vs. 0.43 ± 0.51 mmol L-1  min-1  bar-1  cm-2 , P = 0.003, <0.0001 and <0.0001, respectively). Post-exercise mitochondrial oxidative synthesis rates (a marker of mitochondrial function) tended to be longer in SARC but did not reach significance (17.3 ± 6.4 vs. 14.6 ± 6.5 mmol L-1  min-1 , P = 0.2). However, relative increases in end-exercise ADP in SARC (31.8 ± 9.9 vs. 24.0 ± 7.3 mmol L-1 , P = 0.008) may have been a compensatory mechanism. Mitochondrial complex activity was found to be associated with exercise-induced drops in PCr [citrate synthetase activity (CS), Spearman correlation rho = -0.42, P = 0.03] and end-exercise ADP (complex III, rho = -0.52, P = 0.01; CS rho = -0.45, P = 0.02; SDH rho = -0.45, P = 0.03), with CS also being strongly associated with the PCr recovery rate following low intensity exercise (rho = -0.47, P = 0.02), and the cost of contraction at high intensity (rho = -0.54, P = 0.02). Interestingly, at high intensity, the fractional contribution of oxidative phosphorylation to exercise was correlated with activity in complex II (rho = 0.5, P = 0.03), CS (rho = 0.47, P = 0.02) and SDH (rho = 0.46, P = 0.03), linking increased mitochondrial complex activity with increased ability to generate energy through oxidative pathways. CONCLUSIONS: This study used 31 P MRS to assess ATP utilization and resynthesis in sarcopenic muscle and demonstrated abnormal increases in the energy cost during exercise and perturbed mitochondrial energetics in recovery. Associations between mitochondrial complex activity and the fractional contribution to energy requirement during exercise indicate increased ability to generate energy oxidatively in those with better mitochondrial complex activity.

Postmenopausal Chinese-Singaporean Women Have a Higher Ratio of Visceral to Subcutaneous Adipose Tissue Volume than Caucasian Women of the Same Age and BMI.

Central fat accumulation is a significant determinant of cardio-metabolic health risk, known to differ between ethnically distinct human populations. Despite evidence for preferential central adiposity in Asian populations, the proportional distribution between the subcutaneous and visceral compartments in Chinese postmenopausal women has not been thoroughly investigated. For this analysis, volumetrically quantified subcutaneous and visceral adipose tissue (SAT, VAT) in the pelvic and abdominal regions of postmenopausal Asian (Chinese-Singaporean) and Caucasian (German) women matched for age and Body Mass Index (BMI) was undertaken, to examine such differences between the two groups. Volumes were calculated from segmentations of magnetic resonance imaging datasets of the abdomen and pelvis. Despite SAT, VAT, and the corresponding total adipose tissue (TAT) being similar between the groups, VAT/SAT and VAT/TAT were higher in the Asian group (by 24.5% and 18.2%, respectively, each p = 0.02). Further, VAT/SAT and VAT/TAT were positively correlated with BMI in the Caucasian group only (p = 0.02 and p = 0.01, respectively). We concluded that VAT is proportionally higher in the non-obese Asian women, compared to the Caucasian women of matched age and BMI. This conclusion is in agreement with existing literature showing higher abdominal adiposity in Asian populations. Additionally, in the Asian group, BMI did not correlate with visceral adiposity on a significant level. Further analysis is required to examine the extent to which this increased VAT may impact cardio-metabolic health. There is, however, a need to emphasize healthy lifestyle behaviors in non-obese post-menopausal women of Chinese ancestry.

Folate and Vitamin B-12 Status Is Associated With Bone Mineral Density and Hip Strength of Postmenopausal Chinese-Singaporean Women.

The role of micronutrients such as folate and vitamin B-12 in bone quality has been widely studied with conflicting results. Ethnicity seems to play a large role on nutrient intake, as diet varies across cultures. In this study, we examined the relationships of BMD, proximal femur strength, and bone resorption with plasma folate and vitamin B-12 in a cohort of 93 healthy postmenopausal women of Chinese-Singaporean descent. The parameters examined were areal (aBMD) and volumetric BMD (vBMD) of the proximal femur and the third lumbar vertebra (L3), total body aBMD, proximal femur bending, compressive and impact strength indices (composite strength indices) and circulating levels of C-telopeptide of type I collagen. Eighteen participants (19.4%) had aBMD in the osteoporotic range (osteoporosis group), 59 (63.4%) in the osteopenic range (osteopenia group), and the remaining 16 (17.2%) in the normal range (normal BMD group). Circulating folate levels were significantly higher in the normal BMD group compared with the osteoporosis group. Using linear regression analysis, we found that overall, aBMD and vBMD are positively associated with folate concentrations, whereas composite strength indices were positively associated with vitamin B-12 concentrations. These findings support the existing literature and suggest a link between levels of circulating folate/vitamin B-12 and BMD/bone strength in the cohort examined. Further investigation is needed to examine if individuals with inadequate circulating levels of these nutrients could decrease their risk for fragility fractures through better nutrition or vitamin supplementation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Association of Insulin Resistance with Bone Strength and Bone Turnover in Menopausal Chinese-Singaporean Women without Diabetes.

Insulin resistance (IR) is accompanied by increased areal or volumetric bone mineral density (aBMD or vBMD), but also higher fracture risk. Meanwhile, imbalances in bone health biomarkers affect insulin production. This study investigates the effect of IR on proximal femur and lumbar spine BMD, femoral neck bending, compressive and impact strength indices (Composite Strength Indices) and circulating levels of parathyroid hormone (PTH), C-telopeptide of Type I collagen (CTx-1) and 25(OH) Vitamin D3, in a cohort of 97 healthy, non-obese, menopausal Chinese-Singaporean women. Lumbar spine aBMD was inversely associated with IR and dependent on lean body mass (LBM) and age. No such associations were found for vBMD of the third lumbar vertebra, aBMD and vBMD of the proximal femur, or circulating levels of PTH, CTx-1 and 25(OH) Vitamin D3. Composite Strength Indices were inversely associated with IR and independent of LBM, but after adjusting for fat mass and age, this association remained valid only for the impact strength index. Composite Strength Indices were significantly lower in participants with a high degree of IR. Our findings on IR and Composite Strength Indices relationships were in agreement with previous studies on different cohorts, but those on IR and BMD associations were not.

Configurational Disorder of Water Hydrogen-Bond Network at the Protein Dynamical Transition.

We introduce a novel strategy to quantify the disorder of extended water-water hydrogen-bond (HB) networks sampled in particle-based computer simulations. The method relies on the conformational clustering of the HB connectivity states. We successfully applied it to unveil the fine relationship among the protein dynamical transition in hydrated powder, which marks the activation of protein flexibility at Td ≈ 240 K, and the sudden increase in the configurational disorder of the water HB network enveloping the proteins. Our finding links, in the spirit of the Adam-Gibbs relationship, the diffusivity of protein atoms, as quantified by the hydrogen mean-square displacements, and the thermodynamic solvent configurational entropy.

Stability and Function at High Temperature. What Makes a Thermophilic GTPase Different from Its Mesophilic Homologue.

Comparing homologous enzymes adapted to different thermal environments aids to shed light on their delicate stability/function trade-off. Protein mechanical rigidity was postulated to secure stability and high-temperature functionality of thermophilic proteins. In this work, we challenge the corresponding-state principle for a pair of homologous GTPase domains by performing extensive molecular dynamics simulations, applying conformational and kinetic clustering, as well as exploiting an enhanced sampling technique (REST2). While it was formerly shown that enhanced protein flexibility and high temperature stability can coexist in the apo hyperthermophilic variant, here we focus on the holo states of both homologues by mimicking the enzymatic turnover. We clearly show that the presence of the ligands affects the conformational landscape visited by the proteins, and that the corresponding state principle applies for some functional modes. Namely, in the hyperthermophilic species, the flexibility of the effector region ensuring long-range communication and of the P-loop modulating ligand binding are recovered only at high temperature.

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains.

In this work, we address the question of whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologous G domains of different stability: the mesophilic G domain of the elongation factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-1α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time scale, we show that both proteins host water molecules in internal cavities and that these molecules exchange with the external solution in the nanosecond time scale. The hydration free energy of these sites evaluated via extensive calculations is found to be favorable for both systems, with the hyperthermophilic protein offering a slightly more favorable environment to host water molecules. We estimate that, under ambient conditions, the free energy gain due to internal hydration is about 1.3 kcal/mol in favor of the hyperthermophilic variant. However, we also find that, at the high working temperature of the hyperthermophile, the cavities are rather dehydrated, meaning that under extreme conditions other molecular factors secure the stability of the protein. Interestingly, we detect a clear correlation between the hydration of internal cavities and the protein conformational landscape. The emerging picture is that internal hydration is an effective observable to probe the conformational landscape of proteins. In the specific context of our investigation, the analysis confirms that the hyperthermophilic G domain is characterized by multiple states and it has a more flexible structure than its mesophilic homologue.

Interface matters: the stiffness route to stability of a thermophilic tetrameric malate dehydrogenase.

In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate.

Importance of the ion-pair interactions in the OPEP coarse-grained force field: parametrization and validation.

We have derived new effective interactions that improve the description of ion-pairs in the OPEP coarse-grained force field without introducing explicit electrostatic terms. The iterative Boltzmann inversion method was used to extract these potentials from all atom simulations by targeting the radial distribution function of the distance between the center of mass of the side-chains. The new potentials have been tested on several systems that differ in structural properties, thermodynamic stabilities and number of ion-pairs. Our modeling, by refining the packing of the charged amino-acids, impacts the stability of secondary structure motifs and the population of intermediate states during temperature folding/unfolding; it also improves the aggregation propensity of peptides. The new version of the OPEP force field has the potentiality to describe more realistically a large spectrum of situations where salt-bridges are key interactions.

How conformational flexibility stabilizes the hyperthermophilic elongation factor G-domain.

Proteins from thermophilic organisms are stable and functional well above ambient temperature. Understanding the molecular mechanism underlying such a resistance is of crucial interest for many technological applications. For some time, thermal stability has been assumed to correlate with high mechanical rigidity of the protein matrix. In this work we address this common belief by carefully studying a pair of homologous G-domain proteins, with their melting temperatures differing by 40 K. To probe the thermal-stability content of the two proteins we use extensive simulations covering the microsecond time range and employ several different indicators to assess the salient features of the conformational landscape and the role of internal fluctuations at ambient condition. At the atomistic level, while the magnitude of fluctuations is comparable, the distribution of flexible and rigid stretches of amino-acids is more regular in the thermophilic protein causing a cage-like correlation of amplitudes along the sequence. This caging effect is suggested to favor stability at high T by confining the mechanical excitations. Moreover, it is found that the thermophilic protein, when folded, visits a higher number of conformational substates than the mesophilic homologue. The entropy associated with the occupation of the different substates and the thermal resilience of the protein intrinsic compressibility provide a qualitative insight on the thermal stability of the thermophilic protein as compared to its mesophilic homologue. Our findings potentially open the route to new strategies in the design of thermostable proteins.