Monitoring conformational rearrangements in the substrate-binding site of a membrane transport protein by mass spectrometry

Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low ( approximately 25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding.     

Synthesis and biological evaluation of 5-substituted and 4,5-disubstituted-2-arylamino oxazole TRPV1 antagonists

The synthesis and structure–activity relationships of a series of 5-monosubstituted and 4,5-disubstituted 2-arylaminooxazoles as novel antagonists of the transient receptor potential vanilloid 1 (TRPV1) receptor are described. The 7-hydroxy group of the tetrahydronaphthyl moiety on the 2-amino substituent of the oxazole ring was important for obtaining excellent in vitro potency at the human TRPV1 receptor, while a variety of alkyl and phenyl substituents at the 4- and 5-positions of the oxazole ring were well tolerated and yielded potent TRPV1 antagonists. Despite excellent in vitro potency, the 5-monosubstituted compounds suffered from poor pharmacokinetics. It was found that 4,5-disubstitution on the oxazole ring was critical to the improvement of the overall pharmacokinetic profile of these analogues, which led to the discovery of compound (R)-27, a novel TRPV1 antagonist with good oral activity in preclinical animal models of pain.     

Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.     

An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.     

Lipids including cholesteryl linoleate and cholesteryl arachidonate contribute to the inherent antibacterial activity of human nasal fluid

Mucosal surfaces provide first-line defense against microbial invasion through their complex secretions. The antimicrobial activities of proteins in these secretions have been well delineated, but the contributions of lipids to mucosal defense have not been defined. We found that normal human nasal fluid contains all major lipid classes (in micrograms per milliliter), as well as lipoproteins and apolipoprotein A-I. The predominant less polar lipids were myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid, cholesterol, and cholesteryl palmitate, cholesteryl linoleate, and cholesteryl arachidonate. Normal human bronchioepithelial cell secretions exhibited a similar lipid composition. Removal of less-polar lipids significantly decreased the inherent antibacterial activity of nasal fluid against Pseudomonas aeruginosa, which was in part restored after replenishing the lipids. Furthermore, lipids extracted from nasal fluid exerted direct antibacterial activity in synergism with the antimicrobial human neutrophil peptide HNP-2 and liposomal formulations of cholesteryl linoleate and cholesteryl arachidonate were active against P. aeruginosa at physiological concentrations as found in nasal fluid and exerted inhibitory activity against other Gram-negative and Gram-positive bacteria. These data suggest that host-derived lipids contribute to mucosal defense. The emerging concept of host-derived antimicrobial lipids unveils novel roads to a better understanding of the immunology of infectious diseases.     

Melanoma cell sensitivity to Docetaxel-induced apoptosis is determined by class III beta-tubulin levels

We have previously shown that Docetaxel-induced variable degrees of apoptosis in melanoma. In this report, we studied the beta-tubulin repertoire of melanoma cell lines and show that class III beta-tubulin expression correlated with Docetaxel-resistance. Sensitive cells showed low levels of class III beta-tubulin with little microtubular incorporation, whereas class III beta-tubulin expression was higher in resistant cells and was incorporated into the cytoskeleton. As proof of concept, abrogation of class III by siRNA reverted Docetaxel-resistant cells to a sensitive phenotype, restoring the microtubular polymerisation response and promoting high levels of apoptosis through Bax activation. These results suggest that phenotypic expression of beta-tubulin class III in melanoma may help identify patients with melanoma that can respond to taxanes.