Keywords

Cholera, Circular dichroism, Fluorescence spectroscopy, Membrane lipids, Phospholipase A2, Phospholipids, Proteins, Ricin, Toxins

Abstract

A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A₂ (PLA₂). Lipid thermal phase transition was found to have a dramatic effect on PLA₂ activity. It was also established that although membrane binding and insertion was essential for of PLA₂ activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA₂ secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA₂. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.

Notes

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Graduation Date

2011

Semester

Summer

Advisor

Tatulian, Suren

Degree

Doctor of Philosophy (Ph.D.)

College

College of Graduate Studies

Department

Biomedical Sciences

Format

application/pdf

Identifier

CFE0004035

URL

http://purl.fcla.edu/fcla/etd/CFE0004035

Language

English

Release Date

August 2011

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

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