Sequence and structure comparison of ATP synthase F0 subunits 6 and 8 in notothenioid fish

Mitochondrial changes such as tight coupling of the mitochondria have facilitated sustained oxygen and respiratory activity in haemoglobin-less icefish of the Channichthyidae family. We aimed to characterise features in the sequence and structure of the proteins directly involved in proton transport, which have potential physiological implications. ATP synthase subunit a (ATP6) and subunit 8 (ATP8) are proteins that function as part of the F0 component (proton pump) of the F0F1complex. Both proteins are encoded by the mitochondrial genome and involved in oxidative phosphorylation. To explore mitochondrial sequence variation for ATP6 and ATP8 we analysed sequences from C. gunnari and C. rastrospinosus and compared them with their closely related red-blooded species and eight other vertebrate species. Our comparison of the amino acid sequence of these proteins reveals important differences that could underlie aspects of the unique physiology of the icefish. In this study we find that changes in the sequence of subunit a of the icefish C. gunnari at position 35 where there is a hydrophobic alanine which is not seen in the other notothenioids we analysed. An amino acid change of this type is significant since it may have a structural impact. The biology of the haemoglobin-less icefish is necessarily unique and any insights about these animals will help to generate a better overall understanding of important physiological pathways

The dysregulated Pink1- drosophila mitochondrial proteome is partially corrected with exercise

One of the genes which has been linked to the onset of juvenile/early onset Parkinson’s disease (PD) is PINK1. There is evidence that supports the therapeutic potential of exercise in the alleviation of PD symptoms. It is possible that exercise may enhance synaptic plasticity, protect against neuro-inflammation and modulate L-Dopa regulated signalling pathways. We explored the effects of exercise on Pink1 deficient Drosophila melanogaster which undergo neurodegeneration and muscle degeneration. We used a ‘power-tower’ type exercise platform to deliver exercise activity to Pink1- and age matched wild-type Drosophila. Mitochondrial proteomic profiles responding to exercise were obtained. Of the 516 proteins identified, 105 proteins had different levels between Pink1- and wild-type non-exercised Drosophila. Gene ontology enrichment analysis and STRING network analysis highlighted proteins and pathways with altered expression within the mitochondrial proteome. Comparison of the Pink1- exercised proteome to wild-type proteomes showed that exercising the Pink1- Drosophila caused their proteomic profile to return towards wild-type levels

Mitochondrial Haemoglobin Is Upregulated with Hypoxia in Skeletal Muscle and Has a Conserved Interaction with ATP Synthase and Inhibitory Factor 1

open access articleThe globin protein superfamily has diverse functions. Haemoglobin has been found in non-erythroid locations, including within the mitochondria. Using co-immunoprecipitation and in silico methods, we investigated the interaction of mitochondrial haemoglobin with ATP synthase and its associated proteins, including inhibitory factor 1 (IF1). We measured the expression of mitochondrial haemoglobin in response to hypoxia. In vitro and in silico evidence of interactions between mitochondrial haemoglobin and ATP synthase were found, and we report upregulated mitochondrial haemoglobin expression in response to hypoxia within skeletal muscle tissue. Our observations indicate that mitochondrial pH and ATP synthase activity are implicated in the mitochondrial haemoglobin response to hypoxia

Understanding the role of mitochondrial haemoglobin - an in silico approach

About 60 years ago, Nobel Laureate, Max F. Perutz, who discovered the structure of the globular protein Haemoglobin (Hb), suggested that changes in the molecule were seen in neurodegenerative diseases and argued its role as a drug receptor. Neurodegeneration is more likely to be affected by a fundamental yet inevitable phenomenon of ageing. There has been sufficient evidence that changes in the powerhouse of the cell, mitochondria dysfunction, is one of the hallmarks of ageing and neurodegeneration. Yet we are still far off from deciphering direct links and pathways conclusively leading to it. The project was aimed to find that, if Hb is localised in the mitochondria, it must influence the proteins situated there. Hence to validate the theory the project entailed working on the unique and mysterious icefish that are the only vertebrate organisms without Hb and acted as a model to understand the differences observed in the presence/absence of this protein. In general protein-protein interactions (PPI) can influence many biological processes, metabolic pathways and, cell-to-cell interactions (Braun and Gingras, 2012). These interactions could be transient or permanent (Irene M A Nooren and Thornton, 2003), where transient interactions would form signalling pathways and permanent interactions will form a stable protein complex. New functionality of a characterised protein or a new protein can be predicted on the evidence of their interaction with well characterised proteins in the proteome. PPI data can be categorised into three types, -in vitro, which includes liquid chromatography (LC), gel-electrophoresis, western blots, coimmunoprecipitation, mass spectrophotometry (MS), X-ray crystallographic, NMR techniques, -in vivo, where the techniques are involved with the whole organism, -in silico, techniques involving, sequence and structural based approaches, chromosome proximity, phylogenetic tree, and proteins/gene expression data. The thesis entails in vitro and in silico approaches combined to decipher a Hb interactome. The three result chapters were primarily focussed on important mitochondrial proteins associated with mitochondrial Hb. (i) First, new insights on the ATP synthase mitochondrial encoded Fo motor subunits, ATP6 and ATP8 in the icefish were established using sequential and structural comparison of the nucleotide and protein sequences, where changes were observed in the Hb-less icefish C. gunnari. The changes in protein expression were also observed for ATP6 protein in the icefish using immunoblotting (ii) Second, changes in the mitochondrial proteome of the icefish were observed when compared to closely related red-blooded fish, in two different muscle tissues, red and white, using LC/LC-MS and network analysis. The changes were observed in important pathways such as citric acid cycle, ribosome machinery and fatty acid degradation. (iii) Finally, the last chapter aimed at understanding the sequential and structural changes in the 3’ remnant haemoglobin alpha (HbA) protein in the icefish. A plausible new role for HbA was suggested, where interaction between natural inhibitor of ATP synthase, ATPIF1 and HbA was established using molecular docking and simulations and supported by in vitro co- immunoprecipitation/immunoblotting. In summary the thesis adds onto our understanding of mitochondrial Hb and its putative role in the mitochondria

Understanding the role of mitochondrial haemoglobin - an in silico approach

About 60 years ago, Nobel Laureate, Max F. Perutz, who discovered the structure of the globular protein Haemoglobin (Hb), suggested that changes in the molecule were seen in neurodegenerative diseases and argued its role as a drug receptor. Neurodegeneration is more likely to be affected by a fundamental yet inevitable phenomenon of ageing. There has been sufficient evidence that changes in the powerhouse of the cell, mitochondria dysfunction, is one of the hallmarks of ageing and neurodegeneration. Yet we are still far off from deciphering direct links and pathways conclusively leading to it. The project was aimed to find that, if Hb is localised in the mitochondria, it must influence the proteins situated there. Hence to validate the theory the project entailed working on the unique and mysterious icefish that are the only vertebrate organisms without Hb and acted as a model to understand the differences observed in the presence/absence of this protein. In general protein-protein interactions (PPI) can influence many biological processes, metabolic pathways and, cell-to-cell interactions (Braun and Gingras, 2012). These interactions could be transient or permanent (Irene M A Nooren and Thornton, 2003), where transient interactions would form signalling pathways and permanent interactions will form a stable protein complex. New functionality of a characterised protein or a new protein can be predicted on the evidence of their interaction with well characterised proteins in the proteome. PPI data can be categorised into three types, -in vitro, which includes liquid chromatography (LC), gel-electrophoresis, western blots, coimmunoprecipitation, mass spectrophotometry (MS), X-ray crystallographic, NMR techniques, -in vivo, where the techniques are involved with the whole organism, -in silico, techniques involving, sequence and structural based approaches, chromosome proximity, phylogenetic tree, and proteins/gene expression data. The thesis entails in vitro and in silico approaches combined to decipher a Hb interactome. The three result chapters were primarily focussed on important mitochondrial proteins associated with mitochondrial Hb. (i) First, new insights on the ATP synthase mitochondrial encoded Fo motor subunits, ATP6 and ATP8 in the icefish were established using sequential and structural comparison of the nucleotide and protein sequences, where changes were observed in the Hb-less icefish C. gunnari. The changes in protein expression were also observed for ATP6 protein in the icefish using immunoblotting (ii) Second, changes in the mitochondrial proteome of the icefish were observed when compared to closely related red-blooded fish, in two different muscle tissues, red and white, using LC/LC-MS and network analysis. The changes were observed in important pathways such as citric acid cycle, ribosome machinery and fatty acid degradation. (iii) Finally, the last chapter aimed at understanding the sequential and structural changes in the 3’ remnant haemoglobin alpha (HbA) protein in the icefish. A plausible new role for HbA was suggested, where interaction between natural inhibitor of ATP synthase, ATPIF1 and HbA was established using molecular docking and simulations and supported by in vitro co- immunoprecipitation/immunoblotting. In summary the thesis adds onto our understanding of mitochondrial Hb and its putative role in the mitochondria

Proteomic analysis of the ATP synthase interactome in notothenioids highlights a pathway that inhibits ceruloplasmin production

Antarctic notothenioids have unique adaptations that allow them to thrive in subzero Antarctic waters. Within the suborder Notothenioidei, species of the family Channichthyidae (icefish) lack hemoglobin and in some instances myoglobin too. In studies of mitochondrial function of notothenioids, few have focused specifically on ATP synthase. In this study, we find that the icefish Champsocephalus gunnari has a significantly higher level of ATP synthase subunit α expression than the red-blooded Notothenia rossii, but a much smaller interactome than the other species. We characterize the interactome of ATP synthase subunit α in two red-blooded species Trematomus bernacchii, N. rossii, and in the icefish Chionodraco rastrospinosus and C. gunnari and find that, in comparison with the other species, reactome enrichment for C. gunnari lacks chaperonin-mediated protein folding, and fewer oxidative-stress-associated proteins are present in the identified interactome of C. gunnari. Reactome enrichment analysis also identifies a transcript-specific translational silencing pathway for the iron oxidase protein ceruloplasmin, which has previously been reported in studies of icefish as distinct from other red-blooded fish and vertebrates in its activity and RNA transcript expression. Ceruloplasmin protein expression is detected by Western blot in the liver of T. bernacchii, but not in N. rossii, C. rastrospinosus, and C. gunnari. We suggest that the translation of ceruloplasmin transcripts is silenced by the identified pathway in icefish notothenioids, which is indicative of altered iron metabolism and Fe(II) detoxification

Quantitative Proteomics and Network Analysis of Differentially Expressed Proteins in Proteomes of Icefish Muscle Mitochondria Compared with Closely Related Red-Blooded Species

Antarctic icefish are extraordinary in their ability to thrive without haemoglobin. We wanted to understand how the mitochondrial proteome has adapted to the loss of this protein. Metabolic pathways that utilise oxygen are most likely to be rearranged in these species. Here, we have defined the mitochondrial proteomes of both the red and white muscle of two different icefish species (Champsocephalus gunnari and Chionodraco rastrospinosus) and compared these with two related red-blooded Notothenioids (Notothenia rossii, Trematomus bernacchii). Liquid Chromatography-Mass spectrometry (LC-MS/MS) was used to generate and examine the proteomic profiles of the two groups. We recorded a total of 91 differentially expressed proteins in the icefish red muscle mitochondria and 89 in the white muscle mitochondria when compared with the red-blooded related species. The icefish have a relatively higher abundance of proteins involved with Complex V of oxidative phosphorylation, RNA metabolism, and homeostasis, and fewer proteins for striated muscle contraction, haem, iron, creatine, and carbohydrate metabolism. Enrichment analyses showed that many important pathways were different in both red muscle and white muscle, including the citric acid cycle, ribosome machinery and fatty acid degradation. Life in the Antarctic waters poses extra challenges to the organisms that reside within them. Icefish have successfully inhabited this environment and we surmise that species without haemoglobin uniquely maintain their physiology. Our study highlights the mitochondrial protein pathway differences between similar fish species according to their specific tissue oxygenation idiosyncrasies