Single muscle fiber proteomics reveals unexpected mitochondrial specialization

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high‐sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype‐specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type‐resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.     

Paraoxonase 1 (PON1) polymorphisms, haplotypes and activity in predicting cad risk in North-West Indian Punjabis

Background

Human serum paraoxonase-1 (PON1) prevents oxidation of low density lipoprotein cholesterol (LDL-C) and hydrolyzes the oxidized form, therefore preventing the development of atherosclerosis. The polymorphisms of PON1 gene are known to affect the PON1 activity and thereby coronary artery disease (CAD) risk. As studies are lacking in North-West Indian Punjabi''s, a distinct ethnic group with high incidence of CAD, we determined PON1 activity, genotypes and haplotypes in this population and correlated them with the risk of CAD.

Methodology/Principal Findings

350 angiographically proven (≥70% stenosis) CAD patients and 300 healthy controls were investigated. PON1 activity was determined towards paraoxon (Paraoxonase; PONase) and phenylacetate (Arylesterase; AREase) substrates. In addition, genotyping was carried out by using multiplex PCR, allele specific oligonucleotide –PCR and PCR-RFLP methods and haplotyping was determined by PHASE software. The serum PONase and AREase activities were significantly lower in CAD patients as compared to the controls. All studied polymorphisms except L55M had significant effect on PONase activity. However AREase activity was not affected by them. In a logistic regression model, after adjustment for the conventional risk factors for CAD, QR (OR: 2.73 (1.57–4.72)) and RR (OR, 16.24 (6.41–41.14)) genotypes of Q192R polymorphism and GG (OR: 2.07 (1.02–4.21)) genotype of −162A/G polymorphism had significantly higher CAD risk. Haplotypes L-T-G-Q-C (OR: 3.25 (1.72–6.16)) and L-T-G-R-G (OR: 2.82 (1.01–7.80)) were also significantly associated with CAD.

Conclusions

In conclusion this study shows that CAD patients had lower PONase and AREase activities as compared to the controls. The coding Q192R polymorphism, promoter −162A/G polymorphism and L-T-G-Q-C and L-T-G-R-G haplotypes are all independently associated with CAD.     

Low serum PON1 activity: an independent risk factor for coronary artery disease in North-West Indian type 2 diabetics

Posttranslational modifications of proteins have profound effects on many aspects of their function and have received much attention due to the importance of these processes in epigenetic regulation. In this study, we report that deleted azoospermia associated protein 1 (DAZAP1)/proline-rich RNA binding protein (Prrp), a multifunctional RNA binding protein which is essential for spermatogenesis and normal cell growth, is acetylated at Lysine 150 within its RNA binding domain. The acetylation is predominantly observed in nuclear Prrp, and the nonacetylated form is in cytoplasm. Considering that Prrp is a shuttling protein, we suggest that the acetylation cycle at Prrp K150 regulates nucleocytoplasmic transport in cells.     

Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs

Detergents are indispensable solubilizing agents in the purification and analysis of membrane proteins. For mass spectrometric identification of proteins, it is essential that detergents are removed prior to analysis, necessitating an in-gel digestion step. Here, we report a procedure that allows use of detergents and in-solution digestion of proteins. Crude membrane preparations from mouse brain were solubilized with Triton X-100, CHAPS, or SDS, and the detergents were depleted from the membrane proteins using a desalting column equilibrated with 8 M urea. Following digestion with endoproteinase Lys-C, the resulting peptides were analyzed by LC-MS/MS on Linear ion trap-Orbitrap instrument. Applying stringent identification criteria, in single-LC-MS-runs, 1059 +/- 108 proteins, including 797 +/- 43 membrane proteins, were mapped from mouse brain. The identified proteins represented a broad spectrum of neurotransmitter receptors and other ion channels. The general applicability of the method is demonstrated by profiling of membrane proteins from four other mouse organs. Single-run analyses of eye, liver, spleen, and skeletal muscle allowed identification of 522 +/- 9, 610 +/- 7, 777 +/- 8, and 307 +/- 7 membrane proteins. Our results demonstrate that membrane proteins can be analyzed as efficiently as soluble proteins.     

Benzoxazine derivatives of phytophenols show anti-plasmodial activity via sodium homeostasis disruption

Development of new class of anti-malarial drugs is an essential requirement for the elimination of malaria. Bioactive components present in medicinal plants and their chemically modified derivatives could be a way forward towards the discovery of effective anti-malarial drugs. Herein, we describe a new class of compounds, 1,3-benzoxazine derivatives of pharmacologically active phytophenols eugenol (compound 3) and isoeugenol (compound 4) synthesised on the principles of green chemistry, as anti-malarials. Compound 4, showed highest anti-malarial activity with no cytotoxicity towards mammalian cells. Compound 4 induced alterations in the intracellular Na⁺ levels and mitochondrial depolarisation in intraerythrocytic Plasmodium falciparum leading to cell death. Knowing P-type cation ATPase PfATP4 is a regulator for sodium homeostasis, binding of compound 3, compound 4 and eugenol to PfATP4 was analysed by molecular docking studies. Compounds showed binding to the catalytic pocket of PfATP4, however compound 4 showed stronger binding due to the presence of propylene functionality, which corroborates its higher anti-malarial activity. Furthermore, anti-malarial half maximal effective concentration of compound 4 was reduced to 490?nM from 17.54?µM with nanomaterial graphene oxide. Altogether, this study presents anti-plasmodial potential of benzoxazine derivatives of phytophenols and establishes disruption of parasite sodium homeostasis as their mechanism of action.     

Seasonal and geographical toxicity of Indoxacarb against Helicoverpa armigera and influence of different host plants against Indoxacarb in India, 2005–2007

Indoxacarb, an oxadiazine insecticide, was evaluated for its effectiveness against Helicoverpa armigera collected from selected locations in India. Determination of Indoxacarb efficacy was done using a log-dose probit (LDP) bioassay against third instars collected from cotton ( Gossypium arborium ) fields near Akola, India. Monthly levels of toxicity of Indoxacarb were determined from July 2005 to March 2007. The maximum tolerance level of Indoxacarb was reported for the Amaravati strain (5.09 p.p.m.) and the minimum tolerance level for the Fatehbad strain (0.22 p.p.m.). Seasonal monitoring of Indoxacarb toxicity revealed an increased trend in tolerance from July 2005 to February 2006, which decreased from March 2006. The LC₅₀ of Indoxacarb was 2.71 p.p.m. in July 2005 and 17.14 p.p.m. in February 2006. During 2006–2007, the LC₅₀ was 3.84 p.p.m. at the start of the season and in March 2007 it was 13.51 p.p.m. The minimum LC₅₀ of Indoxacarb was reported for H. armigera larvae fed on Legasca spp. (1.62 p.p.m.) and the maximum LC₅₀ was reported for H. armigera reared on chickpea ( Cicer arietium ) (8.45 p.p.m.). LC₅₀ of 2.73 and 4.56 p.p.m. were reported for H. armigera fed on cotton ( Gossypium arborium ) and pigeonpea ( Cajanus cajan ), respectively.     

Large-scale phosphosite quantification in tissues by a spike-in SILAC method

Despite progress in mass spectrometry (MS)-based phosphoproteomics, large-scale in vivo analyses remain challenging. Here we report a 'spike-in' stable-isotope labeling with amino acids in cell culture (SILAC) methodology using standards derived from labeled mouse liver cell lines, using which we analyzed insulin signaling. With this approach we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment, creating a very large, accurately quantified in vivo phosphoproteome dataset.