Quantitative analysis of proteins of metabolism by reverse phase protein microarrays identifies potential biomarkers of rare neuromuscular diseases

Abstract

BackgroundMuscle diseases have been associated with changes in the expression of proteins involved in energy metabolism. To this aim we have developed a number of monoclonal antibodies against proteins of energy metabolism.MethodsHerein, we have used Reverse Phase Protein Microarrays (RPMA), a high throughput technique, to investigate quantitative changes in protein expression with the aim of identifying potential biomarkers in rare neuromuscular diseases. A cohort of 73 muscle biopsies that included samples from patients diagnosed of Duchenne (DMD), Becker (BMD), symptomatic forms of DMD and BMD in female carriers (Xp21 Carriers), Limb Girdle Muscular Dystrophy Type 2C (LGMD2C), neuronal ceroid lipofuscinosis (NCL), glycogenosis type V (Mc Ardle disease), isolated mitochondrial complex I deficiency, intensive care unit myopathy and control donors were investigated. The nineteen proteins of energy metabolism studied included members of the mitochondrial oxidation of pyruvate, the tricarboxylic acid cycle, β-oxidation of fatty acids, electron transport and oxidative phosphorylation, glycogen metabolism, glycolysis and oxidative stress using highly specific antibodies.ResultsThe results indicate that the phenotype of energy metabolism offers potential biomarkers that could be implemented to refine the understanding of the biological principles of rare diseases and, eventually, the management of these patients.ConclusionsWe suggest that some biomarkers of energy metabolism could be translated into the clinics to contribute to the improvement of the clinical handling of patients affected by rare diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0424-1) contains supplementary material, which is available to authorized users.

Down-regulation of oxidative phosphorylation in the liver by expression of the ATPase inhibitory factor 1 induces a tumor-promoter metabolic state

“…The primary monoclonal antibodies developed in our lab and used in this study were: HSP60 (1:5,000), anti-NADH-9 (1:1,000), anti-β-F1-ATPase (1:20,000), anti-LDH-A (1:1,000) and anti-GAPDH (1:20,000) [ 53 , 54 ]. The monoclonal antibody specifically recognizing the human [ 15 ] and mouse (Molecular Probes) IF1 proteins were used at 1:200 dilution.…”

Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis

“…The major limitation of quantitative RPPA is the availability of specific monoclonal antibodies (mAbs) against the proteins being studied. Figure 1 shows that the mAbs used in this study only recognized a single protein band at the expected molecular weight in human muscle extracts validating their utilization in RPPA techniques [ 11 – 13 ].

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“…In contrast to the findings in muscle of sIBM, the biopsies of DM patients showed an enhanced expression of the mitochondrial protein Hsp60 and IF1 and a very large increase in the glycolytic PKM2. The βF1-ATPase/LDHA ratio, which has been recently described as a potential biomarker of neuromuscular diseases [ 13 ], failed in the discrimination of IMs except for sIBM patients, which showed an increase of the ratio. However, the βF1-ATPase/PKM2 ratio offered a reliable indicator to discriminate DM from any other IM or from control biopsies.…”

“…Protein samples from muscle biopsies were fractionated on SDS–9% PAGE and blotted with anti-β-F1-ATPase (1:1000), anti-Hsp60 (1:1000), anti-GAPDH (1:1000) and anti-PKM2 (1:1000) from [ 11 ], anti-IF1 (1:500) from [ 12 ], anti-LDH-A (1:1000), anti-GPD1 (1:1000) from [ 13 ], anti-PYGM (Abcam, ab88078; 1:1000), anti-PKM1 (Abcam, ab6191-5; 1:1000), anti phospho-PKM2 (Tyr105) (Cell Signaling, #3827; 1:1000), anti-PDHE1α (Invitrogene, 459400; 1:500), anti-phospho-PDHE1α (Ser293) (Abcam, ab92696; 1:1000), anti-PDK1 (Abcam, ab207450; 1:1000) and anti-β-actin (Sigma-Aldrich, A1978; 1:1000). Peroxidase-conjugated anti-mouse IgGs (Nordic Immunology; 1:3000) were used as secondary antibodies.…”

Co-expression Network Approach Reveals Functional Similarities among Diseases Affecting Human Skeletal Muscle

“…Mounting evidence has suggested that the pathological muscle wasting observed in dystrophies (e.g., DMD) might be due to reduced ATP availability required for maintenance of Ca 2+ homeostasis and fiber regeneration (Timpani et al, 2015 ). Bioenergetic pathway enzymes have recently shown to be relevant biomarkers of muscular and neuromuscular disease progression (Santacatterina et al, 2015 ).…”