Understanding the role of mitochondrial haemoglobin - an in silico approach

Katyal, Gunjan (2022) Understanding the role of mitochondrial haemoglobin - an in silico approach. PhD thesis, University of Nottingham.

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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.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Chakrabarti, Lisa
Stoger, Reinhard
Emes, Richard
Keywords: Mitochondrial Biology, Haemoglobin, ATP synthase, Oxidative Phosphorylation, Ageing, Neurodegneration
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID: 68632
Depositing User: Katyal, Gunjan
Date Deposited: 31 Jul 2022 04:41
Last Modified: 31 Jul 2022 04:41
URI: https://eprints.nottingham.ac.uk/id/eprint/68632

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