Date of Award

2025-12-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Marc Cox

Abstract

Prohibitin (PHB) is a highly conserved mitochondrial protein involved in cell cycle regulation, apoptosis, mitochondrial integrity, macrophage activation, cytokine production and many other critical cell functions. The prohibitin complex, formed by Prohibitin-1 (PHB1) and Prohibitin-2 (PHB2), functions as a mitochondrial membrane-associated receptor. Cancer, neurodegenerative disorders, and renal conditions were found to be linked to an irregular PHB expression. Emerging evidence has also highlighted a significant role for PHB in tuberculosis (TB), particularly through its interactions with Mycobacterium tuberculosis (Mtb) virulence determinants. The molecular mechanisms by which PHB influences TB pathogenesis remain poorly understood. Our preliminary studies showed that PHB1 was pulled down with early secretory antigenic target 6kDa (ESAT-6, or EsxA), a key Mtb virulence factor, suggesting PHB1 may serve as a receptor for EsxA to disrupt mitochondrial membrane. To further explore this interaction, the genes encoding PHB1D28 (PHB1 with deletion of the first 28 amino acids) and PHB2D36 (PHB2 with deletion of the first 36 amino acids) were cloned into pTrham6XHisMBP and pMALC2x expression vectors, respectively. The MBP-PHB1D28 and MBP-PDB2D36 proteins were successfully purified in soluble form by using a combination of chromatographic approaches, including immobilized metal affinity chromatography (IMAC), amylose affinity chromatography, and size exclusion chromatography (SEC). The efforts of cleaving MBP tag off the PHB proteins with TEV and Factor Xa were not successful. TEV cleaved MBP-PHB1D28 partly, and the cleaved products could not be effectively separated. Cleavage of MBP-PHB2D36 with Factor Xa resulted in heterogeneous and inefficient proteolysis, rendering the cleavage products unsuitable for downstream applications. The failed cleavage may be due to the oligomerization of the fusion proteins, which limits proteases to get access to the cleavage sites. Therefore, the MBP-PHB fusion proteins were used to test their interaction with EsxA and EsxB by using MBP as a control. SEC was employed to assess these interactions. Either MBP-PHB1D28 or MBP-PHB2D36 was eluted as a single peak, and the mixture of the two proteins at 1:1 molar ratio showed a distinctive peak, suggesting the formation of hetero-oligomeric complex. In contrast, no new peaks were observed when MBP-PHB1D28 or MBP-PHB2D36 were mixed with EsxA, EsxB, or EsxA/B heterodimer, indicating no interaction between PHB proteins with the Mtb virulence factors. Prohibitins are evolutionally conserved and play a major role in preserving mitochondrial structure. Expression, purification, and characterization of PHB1 and PHB2 are crucial steps in understanding their structure and function as well as their roles in TB pathogenesis. Our findings have paved the way of investigating PHB-related multiple diseases featured by mitochondrial disruption.

Language

en

Provenance

Received from ProQuest

File Size

98 p.

File Format

application/pdf

Rights Holder

Zeina Rhaim

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Included in

Biology Commons

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