Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.     

MRI-based assessment of hip joint translations

To better understand movement limitations and, to some extent, the pathogenesis of osteoarthritis, it is important to quantitatively measure femoroacetabular translations to assess if any joint subluxation occurs. In this paper, we aim at measuring hip joint displacements from magnetic resonance images (MRI) based on a surface registration technique. Because this measurement is related to the location of the hip joint center (HJC), we investigate and compare different HJC estimation approaches based on patient-specific 3D bone models. We estimate the HJC based on a simulated circumduction while minimizing inter-articular distance changes. Measurements of femoroacetabular translations during low amplitude abductions ($80$ samples) and extreme flexions ($60$ samples) in female professional dancers, which is a population potentially exposed to femoroactebaluar impingements, do not show any significant subluxation.     

The Grey Mouse Lemur Uses Season-Dependent Fat or Protein Sparing Strategies to Face Chronic Food Restriction

During moderate calorie restriction (CR) the heterotherm Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days (winter; SD40 and SD80, respectively) and long-days (summer; LD40 and LD80, respectively) acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.     

K-fibre minus ends are stabilized by a RanGTP-dependent mechanism essential for functional spindle assembly

Chromosome segregation requires the formation of K-fibres, microtubule bundles that attach sister kinetochores to spindle poles. Most K-fibre microtubules originate around the chromosomes through a non-centrosomal RanGTP-dependent pathway and become oriented with the plus ends attached to the kinetochore and the minus ends focused at the spindle poles. The capture and stabilization of microtubule plus ends at the kinetochore has been extensively studied but very little is known on how their minus-end dynamics are controlled. Here we show that MCRS1 is a RanGTP-regulated factor essential for non-centrosomal microtubule assembly. MCRS1 localizes to the minus ends of chromosomal microtubules and K-fibres, where it protects them from depolymerization. Our data reveal the existence of a mechanism that stabilizes the minus ends of chromosomal microtubules and K-fibres, and is essential for the assembly of a functional bipolar spindle.     

Mathematical modeling of pulmonary tuberculosis therapy: Insights from a prototype model with rifampin

There is a critical need for improved and shorter tuberculosis (TB) treatment. Current in vitro models of TB, while valuable, are poor predictors of the antibacterial effect of drugs in vivo. Mathematical models may be useful to overcome the limitations of traditional approaches in TB research. The objective of this study was to set up a prototype mathematical model of TB treatment by rifampin, based on pharmacokinetic, pharmacodynamic and disease submodels.The full mathematical model can simulate the time-course of tuberculous disease from the first day of infection to the last day of therapy. Therapeutic simulations were performed with the full model to study the antibacterial effect of various dosage regimens of rifampin in lungs.The model reproduced some qualitative and quantitative properties of the bactericidal activity of rifampin observed in clinical data. The kill curves simulated with the model showed a typical biphasic decline in the number of extracellular bacteria consistent with observations in TB patients. Simulations performed with more simple pharmacokinetic/pharmacodynamic models indicated a possible role of a protected intracellular bacterial compartment in such a biphasic decline.This modeling effort strongly suggests that current dosage regimens of RIF may be further optimized. In addition, it suggests a new hypothesis for bacterial persistence during TB treatment.     

Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli

Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.     

Trpm4 Gene Invalidation Leads to Cardiac Hypertrophy and Electrophysiological Alterations

Rationale

TRPM4 is a non-selective Ca²⁺-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.

Objectives

We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 ^-/-) model.

Methods and Results

Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 ^-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4^+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4^-/- mice were smaller than Trpm4^+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4^-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 ^-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4^-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 ^-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca²⁺ or K⁺ currents involved in the repolarizing phase.

Conclusions

TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.     

An analytical approach for estimating fossil record and diversification events in sharks, skates and rays

Background

Modern selachians and their supposed sister group (hybodont sharks) have a long and successful evolutionary history. Yet, although selachian remains are considered relatively common in the fossil record in comparison with other marine vertebrates, little is known about the quality of their fossil record. Similarly, only a few works based on specific time intervals have attempted to identify major events that marked the evolutionary history of this group.

Methodology/Principal Findings

Phylogenetic hypotheses concerning modern selachians’ interrelationships are numerous but differ significantly and no consensus has been found. The aim of the present study is to take advantage of the range of recent phylogenetic hypotheses in order to assess the fit of the selachian fossil record to phylogenies, according to two different branching methods. Compilation of these data allowed the inference of an estimated range of diversity through time and evolutionary events that marked this group over the past 300 Ma are identified. Results indicate that with the exception of high taxonomic ranks (orders), the selachian fossil record is by far imperfect, particularly for generic and post-Triassic data. Timing and amplitude of the various identified events that marked the selachian evolutionary history are discussed.

Conclusion/Significance

Some identified diversity events were mentioned in previous works using alternative methods (Early Jurassic, mid-Cretaceous, K/T boundary and late Paleogene diversity drops), thus reinforcing the efficiency of the methodology presented here in inferring evolutionary events. Other events (Permian/Triassic, Early and Late Cretaceous diversifications; Triassic/Jurassic extinction) are newly identified. Relationships between these events and paleoenvironmental characteristics and other groups’ evolutionary history are proposed.