Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague–Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-¹H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), β-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35–47% lower but monounsaturated fatty acids were 40–52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.     

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.     

Partial Purification and Characterization of a 37 kDa Extracellular Proteinase from Trichophyton vanbreuseghemii

An exocellular proteinase synthesized by the geophilic dermatophyte Trichophyton vanbreuseghemii has been purified and characterized. The fungus obtained from soil in Iran was cultivated in modified Czapek–Dox liquid medium containing 0.1% bacteriological peptone and 1% glucose as the nitrogen and carbon sources. Partial purification of the proteinase was accomplished by (NH₄)₂SO₄ precipitation, followed by ion exchange chromatography. Analysis of the enzyme by SDS-PAGE revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Proteinase activity was optimum at pH 8, but remained high in the range of pH 7–11. Moreover, the partially purified enzyme presented a keratinolytic activity as evidenced by the keratin azure test. The inhibition profile and the good activity of the enzyme towards the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide suggested that it belonged to the chymotrypsin/subtilisin group of serine proteinases. The keratinolytic properties of T. vanbreuseghemii suggest that this fungus may be an alternative for the recycling of industrial keratinic wastes.     

Protein restriction and cancer

Protein restriction without malnutrition is currently an effective nutritional intervention known to prevent diseases and promote health span from yeast to human. Recently, low protein diets are reported to be associated with lowered cancer incidence and mortality risk of cancers in human. In murine models, protein restriction inhibits tumor growth via mTOR signaling pathway. IGF-1, amino acid metabolic programing, FGF21, and autophagy may also serve as potential mechanisms of protein restriction mediated cancer prevention. Together, dietary intervention aimed at reducing protein intake can be beneficial and has the potential to be widely adopted and effective in preventing and treating cancers.     

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.