Biology subjects

Martin, J. L.

Publications and source records attributed to Martin, J. L..

3 recordsLinked to original sources

bioRxiv2018

Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase, PmScsC

Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance - a key virulence trait of the uropathogen. Each protomer of the enzyme has an N-terminal trimerisation stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, we characterise two PmScsC variants, PmScsC{Delta}N and PmScsC{Delta}Linker. PmScsC{Delta}N, is an N-terminally truncated form of the protomer with two helices of the trimerisation stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsC{Delta}N reported here reveals - as expected - a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant PmScsC{Delta}Linker was designed to remove the 11 amino acid linker and we show that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsC{Delta}Linker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentration, whereas PmScsC{Delta}Linker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase our understanding of disulfide isomerase activity, showing how (i) oligomerisation, (ii) spacing between, and (iii) dynamic motion of, catalytic domains in PmScsC all contribute to its native function.

biochemistry↗

bioRxiv2018

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase

DiSulfide Bond (DSB) oxidative folding enzymes are master regulators of virulence localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K12 has a dithiol oxidizing redox relay pair (DsbA/B), a disulfide isomerizing redox relay pair (DsbC/D) and specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these -DsbA1 and -DsbB form a redox relay pair analogous to E. coli DsbA/B. The third enzyme -DsbA2 incorporates a DsbA-like sequence but does not interact with -DsbB. In comparison with other DsbA enzymes, -DsbA2 has [~]50 extra N-terminal residues. The crystal structure of -DsbA2{Delta}N, the N-terminally truncated form in which these residues are removed confirms the DsbA-like nature of this domain. However, -DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. First, -DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase - ie its role is to shuffle rather than introduce disulfide bonds. Moreover, small-angle X-ray scattering of -DsbA2 reveals a homotrimeric arrangement. Our results allow us to draw conclusions about the factors required for functionally equivalent enzymatic activity across structurally diverse protein architectures.

biochemistry↗

bioRxiv2018

Acute Psychological Stress Triggers Circulating Cell-Free Mitochondrial DNA

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, psychological stress triggers elevation in circulating cell- free mtDNA (ccf-mtDNA). Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibit a 2-3-fold increase in ccf-mtDNA, with no change in nuclear DNA levels, establishing the magnitude and specificity to ccf-mtDNA. In cell-based studies, we show that glucocorticoid signaling - a consequence of psychological stress in humans - is sufficient to induce mtDNA extrusion in a time frame consistent with human psychophysiology. Collectively, these findings provide the first evidence that psychological stress induces ccf-mtDNA and implicate glucocorticoid signaling as a trigger for ccf-mtDNA release. Further work is needed to examine the functional significance of psychological stress-induced ccf-mtDNA as a mitokine in humans.

molecular biology↗