The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

In this study, we obtained unprecedented AFM images of the Na,K-ATPase (NKA) pump after being reconstituted into DPPC and DPPC:DPPE liposomes. The mechanical properties observed in the phase images were associated with protrusions correlated to NKA microdomains, which are the darker areas seen in the AFM phase images. Protrusions in the DPPC–NKA proteoliposomes ranged from 38 to 115 nm, with 74 ± 21 nm diameter and 2.1 ± 1.4 nm height. DPPC:DPPE–NKA proteoliposomes showed protrusions from 21 to 78 nm, with 38 ± 16 nm diameter and 0.7 ± 0.5 nm height. We have estimated the presence of annular lipids in the microdomains considering that the areas of the protrusions should contain αβ oligomers and annular phospholipids. For DPPC–NKA proteoliposomes, we hypothesize that 40 phospholipids surround an (αβ)₂ dimer and 46 phospholipids are present for the DPPC:DPPE–NKA proteoliposomes in an αβ monomer. Catalytic activity measurements of both lipid compositions of proteoliposomes harboring NKA provide strong evidence regarding the protein orientation in the biomembrane. AFM data suggest that DPPC–NKA proteoliposomes are also rightside-out protein orientated, where the protrusions have an average height of 2.1 nm, while for DPPC:DPPE–NKA proteoliposomes, the majority of the protein reconstituted should be inside-out orientated, where the protrusions’ average height is 0.5 nm. This result corroborates with the enzymatic analysis, where 61% and 91% of the enzymatic activity was recovered, respectively. Thus, a new application of AFM as a tool for the determination of topological features of protrusions in proteoliposomes has been brought to the scientific community, in addition to revealing the distinct catalytic orientation of enzymes present in the biomembranes model.

Extracellular vesicles (EVs) are lipid bilayer‐enclosed nanosized particles released by all cell types during physiological as well as pathophysiological processes to carry out diverse biological functions, including acting as sources of cellular dumping, signalosomes and mineralisation nanoreactors. The ability of EVs to perform specific biological functions is due to their biochemical machinery. Among the components of the EVs’ biochemical machinery, surface proteins are of critical functional significance as they mediate the interactions of EVs with components of the extracellular milieu, the extracellular matrix and neighbouring cells. Surface proteins are thought to be native, that is, pre‐assembled on the EVs’ surface by the parent cells before the vesicles are released. However, numerous pieces of evidence have suggested that soluble proteins are acquired by the EVs’ surface from the extracellular milieu and further modulate the biological functions of EVs during innate and adaptive immune responses, autoimmune disorders, complement activation, coagulation, viral infection and biomineralisation. Herein, we will describe the methods currently used to identify the EVs’ surface proteins and discuss recent knowledge on the functional relevance of the soluble proteins acquired by EVs.

Mineralizing cells release a special class of extracellular vesicles known as matrix vesicles (MV), crucial for bone mineralization. Following their release, MV anchor to the extracellular matrix (ECM), where their highly specialized enzymatic machinery facilitates the formation of seed mineral within the MV’s lumen, subsequently releasing it onto the ECM. However, how MV propagate mineral onto the collagenous ECM remains unclear. In this study, we address these questions by exploring the “protein corona” paradigm whereby nanoparticles entering a biological milieu become cloaked by a corona of soluble proteins modifying their biological functions. We isolated native MV from the growth plates of chicken embryos. After removing the protein corona from the native MV using high ionic strength buffer, we obtained shaved MV. Reconstituted MVs were produced by incubating shaved MV with the removed protein corona constituents. Our results show that both the removal and reconstitution of protein corona significantly affect the biochemical and physicochemical properties of MV, resulting in 3 well-defined groups. Shaved MV exhibited an increase in tissue nonspecific alkaline phosphatase (TNAP) activity and a decrease in mineral deposition compared to native MV. Reconstituted MV partially recovered these functions, showing a reduction of TNAP activity and mineral deposition compared to native MV. Furthermore, changes in the protein corona affect the MV ability to anchor to the collagenous ECM, which is crucial for initiating the propagation of the mineral phase within this organic matrix. Proteomic analyses revealed changes in the protein profile of the MV resulting from the removal of the protein corona, indicating that shaved proteins were primarily related to external structural and ECM organization and catabolism. These findings underscore the role of the protein corona in modulating the mineralization capabilities of MV. Understanding these interactions could lead to new therapeutic strategies for enhancing bone repair and regeneration.