Biology subjects

Mackay, C. L.

Publications and source records attributed to Mackay, C. L..

4 recordsLinked to original sources

bioRxiv2026

Localisation of corticosteroids in male mouse kidney by mass spectrometry imaging

Renal sodium balance is important for blood pressure homeostasis and is regulated by corticosteroids, chiefly aldosterone but also glucocorticoids. Abundance of these hormones in functional subregions of the kidney is unknown, previous work being limited to measurements in plasma and urine, and in microdialysate from kidney medulla. Here mass spectrometry imaging (MSI) was applied to map corticosteroids in kidney to understand how their distribution overlays with functional targets. Male C57BL6 mice, aged 10 weeks, were fed diets containing 0.03%, 0.3% or 3% w:w sodium for two weeks and kidneys harvested at cull. Steroids were localised as Girard T derivatives on cryosections using Matrix assisted laser desorption ionisation Fourier transform ion cyclotron MSI, with confirmation by liquid extraction surface analysis. Co-registration was performed on sections stained with hematoxylin and eosin. Corticosterone localised along the papilla, medulla and inner cortex, whereas 11-dehydrocorticosterone was concentrated in the medulla. Higher amounts of aldosterone were present in medulla and outer cortex. Distribution patterns were unchanged by dietary salt, but amounts of corticosterone were elevated, particularly in outer cortex with low salt diet. MSI holds great promise to dissect corticosteroid signalling in functional zones of the kidney.

biochemistry↗

bioRxiv2021

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment revealed by Cryo-EM and hydrogen/deuterium exchange mass spectrometry

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications. TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible.

biochemistry↗

bioRxiv2019

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this proposed iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site.

biochemistry↗

bioRxiv2018

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

Iron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised the Rhodospirillum rubrum EncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the same distinct annular decamer topology observed in R. rubrum EncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass spectrometr analyses show that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The catalytic role of the EncFtn proteins was confirmed by biochemical assays, and we show that Zn(II) ions inhibit the ferroxidase activity of the EncFtn proteins to varying degrees. Our results represent a further step in the characterisation of the recently discovered EncFtn ferritin-like sub-family, indicating a common structural organisation and catalytic activity, despite diverse host environments.

biochemistry↗