Repository logo
 

Investigating the role of mitochondria in the hypoxia response using in vitro and in vivo models


Loading...
Thumbnail Image

Type

Thesis

Change log

Authors

Al-Habib, Alhasan 

Abstract

Human coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) catalyses disulfide bond formation in substrate proteins, mediating their import from the cytoplasm into the mitochondria. CHCHD4 is critical for maintaining intracellular oxygenation and metabolism in human cells, partly by controlling the expression of respiratory chain complex subunits. In cancer, CHCHD4 is required for tumour growth in vivo, with overexpression in a range of cancers correlating with increased tumour progression, the hypoxia gene signature, and poorer patient survival. CHCHD4 (MIA40) is highly evolutionarily conserved and essential in yeast, mice and zebrafish. This thesis explores the role of CHCHD4 in regulating mitochondrial function and hypoxia signalling in vivo, using CRISPR/Cas9 chchd4¬-targeted zebrafish, and in vitro, using human cell systems including patient-derived cells. Zebrafish Chchd4 exists as two paralogues, Chchd4a and Chchd4b; both contain the evolutionarily conserved cysteine motifs required for mitochondrial localisation and function of CHCHD4. I confirmed that chchd4a is essential in zebrafish, with chchd4a-/- fish not surviving to adulthood. In embryos, chchd4a was shown to be required for the expression of a panel of respiratory chain proteins known to be regulated by CHCHD4, unlike chchd4b. In response to hypoxia, compared to wild type (wt), chchd4b-/- embryos exhibited a time-dependent greater induction of Hif-1α protein, yet also a correlative reduction in expression of the Hif-1α target gene egln3. Chchd4b-/- embryos also hatched faster than wt, a process that has been shown by others to coincide with increased Hif-α expression. The accelerated hatching rate of chchd4b-/- embryos was found to be rescued by chchd4a loss in normoxia. To further investigate the functions of the zebrafish Chchd4 paralogues in a cellular context, human U2OS clonal cell lines stably expressing either vector control, chchd4a, or chchd4b cDNA were generated. U2OS-Chchd4a cells exhibited a greater upregulation of HIF-1α and EGLN3 expression in hypoxia compared to the vector control, whereas U2OS-Chchd4b cells showed the opposite. Notably, expression of Chchd4b appeared to be less well tolerated in U2OS cells, whilst also appearing to localise predominantly in the cytoplasm. Taken together, these data indicate that Chchd4a and Chchd4b have distinct functions. Skin fibroblasts isolated from a patient identified to exhibit compound heterozygous mutations in CHCHD4 were also evaluated. Compared to skin fibroblasts from a healthy control patient, the patient-derived mutant CHCHD4 cells exhibited decreased CHCHD4 levels, significantly reduced basal cellular oxygen consumption rate (OCR) and increased extracellular acidification rate (ECAR), as well as reduced expression of MTCO2, a complex IV subunit. Collectively, these data suggest altered mitochondrial function in the CHCHD4 mutant patient-derived cells. In summary, this thesis explores the link between CHCHD4 and HIF signalling in vitro and in vivo, providing novel insight into CHCHD4 function using zebrafish models and human cell systems. The data presented in this thesis show unique and overlapping functions of the Chchd4 paralogues in zebrafish and, for the first time, describe the functional consequences of CHCHD4 mutations identified in a patient.

Description

Date

2022-05-01

Advisors

Ashcroft, Margaret

Keywords

CHCHD4, hypoxia, HIF, disulfide relay, MIA40

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
Sackler Scholarship