Development of cell-free platforms for discovering, characterizing, and engineering post-translational modifications.

Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTM installing proteins often suffer from low-throughput experimental techniques. Here we describe a generalizable, workflow coupling cell-free protein synthesis (CFPS) with AlphaLISA for the rapid expression and testing of PTM installing proteins.

Loss of mitochondria long-chain fatty acid oxidation impairs skeletal muscle contractility by disrupting myofibril structure and calcium homeostasis.

Objective: Abnormal lipid metabolism in mammalian tissues can be highly deleterious, leading to organ failure. Carnitine Palmitoyltransferase 2 (CPT2) deficiency is an inherited metabolic disorder affecting the liver, heart, and skeletal muscle due to impaired mitochondrial oxidation of long-chain fatty acids (mLCFAO) for energy production.

Methods: However, the basis of tissue damage in mLCFAO disorders is not fully understood.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic -GlcNAcylation.

-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human -linked glycans is the difficulty of installing a single -acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (-GlcNAcylation). Here, we develop an method for -GlcNAcylating proteins using the oligosaccharyltransferase PglB from .

Lipid metabolism in dopaminergic neurons influences light entrainment.

Dietary lipids, particularly omega-3 polyunsaturated fatty acids, are speculated to impact behaviors linked to the dopaminergic system, such as movement and control of circadian rhythms. However, the ability to draw a direct link between dopaminergic omega-3 fatty acid metabolism and behavioral outcomes has been limited to the use of diet-based approaches, which are confounded by systemic effects. Here, neuronal lipid metabolism was targeted in a diet-independent manner by manipulation of long-chain acyl-CoA synthetase 6 (ACSL6) expression.

Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.

The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6-/-) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6-/- brains contained lower DHA compared with controls across the life span.

Acyl-CoA synthetases as regulators of brain phospholipid acyl-chain diversity.

Each individual cell-type is defined by its distinct morphology, phenotype, molecular and lipidomic profile. The importance of maintaining cell-specific lipidomic profiles is exemplified by the numerous diseases, disorders, and dysfunctional outcomes that occur as a direct result of altered lipidome. Therefore, the mechanisms regulating cellular lipidome diversity play a role in maintaining essential biological functions.

Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse.

Docosahexaenoic acid (DHA) is an ω-3 dietary-derived polyunsaturated fatty acid of marine origin enriched in testes and necessary for normal fertility, yet the mechanisms regulating the enrichment of DHA in the testes remain unclear. Long-chain ACSL6 (acyl-CoA synthetase isoform 6) activates fatty acids for cellular anabolic and catabolic metabolism by ligating a CoA to a fatty acid, is highly expressed in testes, and has high preference for DHA. Here, we investigated the role of ACSL6 for DHA enrichment in the testes and its requirement for male fertility.

Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform.

Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease.

Acyl-CoA synthetase 6 enriches the neuroprotective omega-3 fatty acid DHA in the brain.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is highly abundant in the brain and confers protection against numerous neurological diseases, yet the fundamental mechanisms regulating the enrichment of DHA in the brain remain unknown. Here, we have discovered that a member of the long-chain acyl-CoA synthetase family, Acsl6, is required for the enrichment of DHA in the brain by generating an Acsl6-deficient mouse (Acsl6). Acsl6 is highly enriched in the brain and lipid profiling of Acsl6 tissues reveals consistent reductions in DHA-containing lipids in tissues highly abundant with Acsl6.

Ceftaroline is active against heteroresistant methicillin-resistant Staphylococcus aureus clinical strains despite associated mutational mechanisms and intermediate levels of resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important infectious human pathogen responsible for diseases ranging from skin and soft tissue infections to life-threatening endocarditis. β-Lactam resistance in MRSA involves acquisition of penicillin-binding protein 2a (PBP2a), a protein with low affinity for β-lactams that mediates cell wall assembly when the normal staphylococcal PBPs (PBP1 to -4) are blocked by these agents. Many MRSA strains display heterogeneous expression of resistance (HeR) against β-lactam antibiotics.

TCA cycle-mediated generation of ROS is a key mediator for HeR-MRSA survival under β-lactam antibiotic exposure.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major multidrug resistant pathogen responsible for several difficult-to-treat infections in humans. Clinical Hetero-resistant (HeR) MRSA strains, mostly associated with persistent infections, are composed of mixed cell populations that contain organisms with low levels of resistance (hetero-resistant HeR) and those that display high levels of drug resistance (homo-resistant HoR). However, the full understanding of β-lactam-mediated HeR/HoR selection remains to be completed.