Abdul Jaleel

In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial proteins

Skeletal muscle mitochondrial dysfunction occurs in many conditions including aging and insulin resistance, but the molecular pathways of the mitochondrial dysfunction remain unclear. Presently, no methodologies are available to measure synthesis rates of individual mitochondrial proteins, which limits our ability to fully understand the translational regulation of gene transcripts. Here, we report a methodology to measure synthesis rates of multiple muscle mitochondrial proteins, which, along with large-scale measurements of mitochondrial gene transcripts and protein concentrations, will enable us to determine whether mitochondrial alteration is due to transcriptional or translational changes. The methodology involves in vivo labeling of muscle proteins with l-[ring-(13)C(6)]phenylalanine, protein purification by two-dimensional gel electrophoresis of muscle mitochondrial fraction, and protein identification and stable isotope abundance measurements by tandem mass spectrometry. Synthesis rates of 68 mitochondrial and 23 nonmitochondrial proteins from skeletal muscle mitochondrial fraction showed a 10-fold range, with the lowest rate for a structural protein such as myosin heavy chain (0.16 +/- 0.04%/h) and the highest for a mitochondrial protein such as dihydrolipoamide branched chain transacylase E2 (1.5 +/- 0.42%/h). This method offers an opportunity to better define the translational regulation of proteins in skeletal muscle or other tissues.

Supramolecular Functionalization and Concomitant Enhancement in Properties of Au25 Clusters

We present a versatile approach for tuning the surface functionality of an atomically precise 25 atom gold cluster using specific host-guest interactions between β-cyclodextrin (CD) and the ligand anchored on the cluster. The supramolecular interaction between the Au25 cluster protected by 4-(t-butyl)benzyl mercaptan, labeled Au25SBB18, and CD yielding Au25SBB18∩CDn (n = 1, 2, 3, and 4) has been probed experimentally using various spectroscopic techniques and was further analyzed by density functional theory calculations and molecular modeling. The viability of our method in modifying the properties of differently functionalized Au25 clusters is demonstrated. Besides modifying their optoelectronic properties, the CD moieties present on the cluster surface provide enhanced stability and optical responses which are crucial in view of the potential applications of these systems. Here, the CD molecules act as an umbrella which protects the fragile cluster core from the direct interaction with many destabilizing agents such as metal ions, ligands, and so on. Apart from the inherent biocompatibility of the CD-protected Au clusters, additional capabilities acquired by the supramolecular functionalization make such modified clusters preferred materials for applications, including those in biology.

Identification of de novo synthesized and relatively older proteins: accelerated oxidative damage to de novo synthesized apolipoprotein A-1 in type 1 diabetes.

OBJECTIVE The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is reported here and can also be applied to other specific proteins. RESEARCH DESIGN AND METHODS To label newly synthesized proteins, [ring-13C6]phenylalanine was intravenously infused for 8 h in type 1 diabetic participants (n = 7) during both insulin treatment and 8 h of insulin deprivation and in nondiabetic participants (n = 7). ApoA-1 isoforms were purified by two-dimensional gel electrophoresis (2DGE) and assessment of protein identity, PTM, and [ring-13C6]phenylalanine isotopic enrichment (IE) was performed by tandem mass spectrometry. RESULTS Five isoforms of plasma ApoA-1 were identified by 2DGE including ApoA-1 precursor (pro-ApoA-1) that contained the relatively highest IE, whereas the older forms contained higher degrees of damage (carbonylation, deamidation) and far less IE. In type 1 diabetes, the relative ratio of IE of [ring-13C6]phenylalanine in an older isoform versus pro-ApoA-1 was higher during insulin deprivation, indicating that de novo synthesized pro-ApoA-1 more rapidly accumulated damage, converting to mature ApoA-1. CONCLUSIONS We developed a mass spectrometry–based methodology to identify the relative age of protein isoforms. The results demonstrated accelerated oxidative damage to plasma ApoA-1, thus offering a potential mechanism underlying the impact of poor glycemic control in type 1 diabetic patients that affects a patients risk for vascular disease.

Differential effects of insulin deprivation and systemic insulin treatment on plasma protein synthesis in type 1 diabetic people

It remains to be determined whether systemic insulin replacement normalizes synthesis rates of different plasma proteins and whether there are differential effects on various plasma proteins. We tested a hypothesis that insulin deprivation differentially affects individual plasma protein synthesis and that systemic insulin treatment may not normalize synthesis of all plasma proteins. We measured synthesis rates of 41 plasma proteins in seven each of type 1 diabetic (T1DM) and nondiabetic participants (ND) using [ring-(13)C(6)]phenylalanine as a tracer. T1DM were studied while on chronic insulin treatment and during 8 h insulin deprivation. Insulin treatment normalized glucose levels, but plasma insulin levels were higher during insulin treatment than during insulin deprivation in T1DM and ND. Individual plasma proteins were purified by affinity chromatography and two-dimensional gel electrophoresis. Only 41 protein gel spots from over 300 were chosen based on their protein homogeneity. Insulin deprivation and hyperglycemia either significantly increased (n = 12) or decreased (n = 12) synthesis rates of 24 of 41 plasma proteins in T1DM compared with ND. Insulin treatment normalized synthesis rates of 13 of these 24 proteins, which were altered during insulin deprivation. However, insulin treatment significantly altered the synthesis of 14 additional proteins. In conclusion, acute insulin deprivation caused both a decrease and increase in synthesis rates of many plasma proteins with various functions. Moreover, chronic systemic insulin treatment not only did not normalize synthesis of all plasma proteins but also altered synthesis of several additional proteins that were unaltered during insulin deprivation.

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Waynes dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.

Comparison of different mass spectrometry techniques in the measurement of L-[ring-13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L-[ring-(13)C(6)]phenylalanine and a bolus dose of L-[ring-(13)C(6)]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC-combustion-isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring-(13)C(6)]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R(2)= 0.9962 and R(2) = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-(13)C(6)]phenylalanine tracer enrichment in low abundance and in small quantity samples.

High throughput proteomic analysis and a comparative review identify the nuclear chaperone, Nucleophosmin among the common set of proteins modulated in Chikungunya virus infection

Abstract Global re-emergence of Chikungunya virus (CHIKV) has renewed the interest in its cellular pathogenesis. We subjected CHIKV-infected Human Embryo Kidney cells (HEK293), a widely used cell-based system for CHIKV infection studies, to a high throughput expression proteomics analysis by Liquid Chromatography–tandem mass spectrometry. A total of 1047 differentially expressed proteins were identified in infected cells, consistently in three biological replicates. Proteins involved in transcription, translation, apoptosis and stress response were the major ones among the 209 proteins that had significant up-regulation. In the set of 45 down-regulated proteins, those involved in carbohydrate and lipid metabolism predominated. A STRING network analysis revealed tight interaction of proteins within the apoptosis, stress response and protein synthesis pathways. We short-listed a common set of 30 proteins that can be implicated in cellular pathology of CHIKV infection by comparing our results and results of earlier CHIKV proteomics studies. Modulation of eight proteins selected from this set was re-confirmed at transcript level. One among them, Nucleophosmin, a nuclear chaperone, showed temporal modulation and cytoplasmic aggregation upon CHIKV infection in double immunofluorescence staining and confocal microscopy. The short-listed cellular proteins will be potential candidates for targeted study of the molecular interactions of CHIKV with host cells. Biological significance Chikungunya remained as a neglected tropical disease till its re-emergence in 2005 in the La RéUnion islands and subsequently, in India and many parts of South East Asia. These and the epidemics that followed in subsequent years ran an explosive course leading to extreme morbidity and attributed mortality to this originally benign virus infection. Apart from classical symptoms of acute fever and debilitating polyarthralgia lasting for several weeks, a number of complications were documented. These included aphthous-like ulcers and vesiculo-bullous eruptions on the skin, hepatic involvement, central nervous system complications such as encephalopathy and encephalitis, and transplacental transmission. The disease has recently spread to the Americas with its initial documentation in the Caribbean islands. The Asian genotype of this positive-stranded RNA virus of the Alphavirus genus has been attributed in these outbreaks. However, the disease ran a similar course as the one caused by the East, Central and South African (ECSA) genotype in the other parts of the world. Studies have documented a number of mutations in the re-emerging strains of the virus that enhances mosquito adaptability and modulates virus infectivity. This might support the occurrence of fiery outbreaks in the absence of herd immunity in affected population. Several research groups work to understand the pathogenesis of chikungunya and the mechanisms of complications using cellular and animal models. A few proteomics approaches have been employed earlier to understand the protein level changes in the infected cells. Our present study, which couples a high throughput proteomic analysis and a comparative review of these earlier studies, identifies a few critical molecules as hypothetical candidates that might be important in this infection and for future study.

Insulin antibody response to bovine insulin therapy : functional significance among insulin requiring young diabetics in India

The majority of young diabetics in India prefer to use low-cost bovine insulin for economic reasons. Therefore, the question of insulin antibody response to bovine insulin and its functional significance is still relevant in the Indian context. We assessed insulin antibody response in 52 young diabetics (type 1, n=25, malnutrition modulated form of diabetes, n=19 and fibrocalculous pancreatopathy (FCP) n=8) on bovine insulin therapy (mean duration 3.0+/-2.1 years) using an internationally standardised in-house radioligand assay. The functional significance of insulin antibody was assessed by calculating their affinity constant, maximum binding capacity and total insulin binding power by Scatchard analysis (type 1, n=14, malnutrition modulated form of diabetes, n=11). All the patients treated with bovine insulin showed high titers of insulin antibodies with S.D. score ranging from 5.1 to 42.0. No significant difference was observed in the mean S.D. score of insulin antibodies in the three diabetic groups. The mean daily insulin dose, maximum insulin binding capacity and total insulin binding power were significantly higher in type 1 when compared to the malnutrition modulated form of diabetes (36+/-8 vs. 26+/-11 IU/day, P<0.05; 9. 7+/-7.8 vs. 4.0+/-3.9 nmol/l, P=0.03 and 59+/-29 vs. 29+/-43, P=0.01, respectively). Insulin antibodies S.D. score and its affinity did not show significant relationship with daily insulin dose and glycemic control (HbAl) at admission. Only 24+/-7% variations in daily insulin requirement were accounted for by total insulin binding power. There was a significant inverse relationship between insulin antibody S.D. score and duration of insulin therapy (r=-0. 4172, P<0.0004). To conclude, insulin antibody response following bovine insulin therapy is not different among type 1, malnutrition modulated form of diabetes and FCP diabetes. The insulin antibody response to bovine insulin therapy does not contribute significantly to increase in daily insulin requirement in bovine insulin treated insulin requiring young diabetics.

Proteome profile of a pandemic Vibrio parahaemolyticus SC192 strain in the planktonic and biofilm condition

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors’ knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.

Impact of Type 1 Diabetes and Insulin Treatment on Plasma Levels and Fractional Synthesis Rate of Retinol-Binding Protein 4

CONTEXT Retinol binding protein 4 (RBP4) levels are elevated in insulin-resistant states and reduced in type 1 diabetes (T1D), but it is unknown whether changes in insulin levels and glycemic control alter RBP4 levels. In vivo synthesis rates of RBP4 and their relationship to RBP4 levels remain to be determined. OBJECTIVE The aim of the study was to determine whether the synthesis rate of RBP4 is altered in people with T1D during both insulin deficiency and insulin treatment. DESIGN Seven T1D participants were studied on two occasions, during 8 h of insulin deprivation and during insulin treatment, and compared with nondiabetic (ND) controls. MAIN OUTCOME MEASURES We measured in vivo fractional synthesis rate of RBP4 using [ring-(13)C(6)]phenylalanine as a tracer and RBP4 concentration in plasma by nephelometric assay and Western blot analyses. RESULTS Plasma RBP4 levels were lower (P < 0.01) in insulin-treated T1D than in ND but were not different between insulin-deprived T1D and ND participants. Synthesis rates of RBP4 in ND (2.46 +/- 0.29%/h) were higher than in insulin-treated T1D (1.45 +/- 0.21) (P = 0.02), but there was no difference between ND and insulin-deprived T1D (2.24 +/- 0.24). Glucose levels were not different between ND and insulin-treated T1D, but insulin levels were higher in insulin-treated T1D (82.8 +/- 2 pmol/liter) than in ND (28.7 +/- 6) and insulin-deprived T1D (4.6 +/- 1.6) (P < 0.01). CONCLUSIONS Insulin treatment that achieved normoglycemia but relative hyperinsulinemia was associated with lower RBP4 synthesis and levels in T1D. Short-term insulin deprivation and hyperglycemia had no effect on RBP4 levels and synthesis rates in T1D.