Biology subjects

Hadziselimovic, A.

Publications and source records attributed to Hadziselimovic, A..

3 recordsLinked to original sources

bioRxiv2021

Delivery of recombinant SARS-CoV-2 envelope protein into human cells

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of "Trojan Horse" anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

biochemistry↗

bioRxiv2020

The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimers disease. The cleavage of APP by {beta}-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by {gamma}-secretase to yield amyloid-{beta} (A{beta}) peptides are essential steps in this pathway. Biochemical evidence suggests amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid ordered phase membrane raft domains. However, direct evidence that C99 preferentially associates with rafts has remained elusive. Here, we test this idea by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles. We find that C99 is essentially excluded from ordered domains in HeLa cells, SH-SY5Y cells and neurons, instead exhibiting a strong (roughly 90%) affinity for disordered domains. The strong association of C99 with disordered domains occurs independently of its cholesterol binding activity, homodimerization, or the familial Alzheimer disease Arctic mutation. Finally, we confirm previous studies suggesting that C99 is processed in the plasma membrane by -secretase, in addition to the well-known {gamma}-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying either that amyloidogenic processing of the protein occurs in disordered regions of the membrane, that processing involves a marginal sub-population of C99 found in rafts, or that as-yet-unidentified protein-protein interactions involving C99 in living cells drive it into rafts to promote its cleavage therein.

neuroscience↗

bioRxiv2017

Mechanisms of KCNQ1 Channel Dysfunction in Long QT Syndrome Involving Voltage Sensor Domain Mutations

Loss-of-function (LOF) mutations in human KCNQ1 are responsible for susceptibility to a life-threatening heart rhythm disorder, the congenital long-QT syndrome (LQTS). Hundreds of KCNQ1 mutations have been identified, but the molecular mechanisms responsible for impaired function are poorly understood. Here, we investigated the impact of 51 KCNQ1 variants located within the voltage sensor domain (VSD), with an emphasis on elucidating effects on cell surface expression, protein folding and structure. For each variant, the efficiency of trafficking to the plasma membrane, the impact of proteasome inhibition, and protein stability were assayed. The results of these experiments, combined with channel functional data, provided the basis for classifying each mutation into one of 6 mechanistic categories. More than half of the KCNQ1 LOF mutations destabilize the structure of the VSD, resulting in mistrafficking and degradation by the proteasome, an observation that underscores the growing appreciation that mutation-induced destabilization of membrane proteins may be a common human disease mechanism. Finally, we observed that 5 of the folding-defective LQTS mutants are located in the VSD S0 helix, where they interact with a number of other LOF mutation sites in other segments of the VSD. These observations reveal a critical role for the S0 helix as a central scaffold to help organize and stabilize the KCNQ1 VSD and, most likely, the corresponding domain of many other ion channels.\n\nOne Sentence SummaryLong QT syndrome-associated mutations in KCNQ1 most often destabilize the protein, leading to mistrafficking and degradation.

biochemistry↗