Biology subjects

Sykes, A.

Publications and source records attributed to Sykes, A..

4 recordsLinked to original sources

bioRxiv2025

Early-stage determinants of T1-S1 conformations in Kv1.3 channels

Early-stage biogenesis of voltage-gated potassium channels (Kv) remains remarkably understudied yet is key to defining co- and post-translational acquisition of Kv secondary, tertiary, and quaternary conformations. Thus, we have studied nascent folding events and their determinants in Kv1.3, a tetrameric ion channel highly expressed in nerve and immune cells. We explore how folding of T1, an intersubunit recognition domain that ensures correct isoform assembly, is modulated by molecular determinants in the Kv subunit. Specifically, we focus on the T1-S1 linker and its highly conserved C-terminal sequence, S0. Using pegylation, a mass-tagging strategy, inter- and intrasubunit crosslinking, we define the molecular determinants of T1-S1 linker accessibility and location. Our findings show that i) dynamic protein-lipid and protein-protein linker interfaces exist, ii) the presence of a T1 domain and its conformation (monomer versus tetramer) impact linker properties, iii) helical formation of S0 occurs in early biogenic stages and does not require the presence of membranes, iv) a core recognition domain (T2) increases T1 dimerization efficiency and promotes tetramer formation, and v) as few as 12 native linker residues enable T1 tetramerization. These findings differ from canonical models and suggest that domain plasticity for Kv1.3 contributes to biogenesis and may underlie domain-domain communication in Kv function.

physiology↗

bioRxiv2024

Parkinson's Disease-Linked Kir4.2 Mutation R28C Leads to Loss of Ion Channel Function

Parkinsons disease (PD) is a complex, progressive neurodegenerative disorder driven by multiple pathogenetic factors, including oxidative stress, mitochondria dysfunction, neuroinflammation, and ion imbalance. Recent evidence highlights the significant role of potassium channels in the pathophysiology of PD. We recently identified a PD-linked genetic mutation in the KCNJ15 gene (KCNJ15p.R28C), encoding the inwardly rectifying potassium channel Kir4.2, within a four-generation family with familial PD. However, the role of the Kir4.2 channel in neurodegenerative diseases remains largely unexplored. This study aimed to elucidate the impact of the KCNJ15p.R28C (Kir4.2R28C) mutation on the biophysical and biochemical properties of Kir4.2. Employing Kir4.2-overexpressing HEK293T cells as a model, we investigated how the mutation affects the channels functional properties, total protein expression, intracellular processing in the endoplasmic reticulum and lysosomes, and plasma membrane trafficking. Patch clamp studies revealed that the Kir4.2R28C mutation results in loss of channel function with significant dominant-negative effects. This dysfunction is partially attributed to the substantial reduction in overall mutant channel protein expression compared to the wild-type (Kir4.2WT). We observed that both Kir4.2WT and Kir4.2R28C proteins undergo glycosylation during the post-translational modification process, albeit with differing protein turnover efficiencies. Furthermore, the Kir4.2R28C mutant exhibits reduced stability and compromised plasma membrane trafficking capacity compared to Kir4.2WT. These findings suggest that the Kir4.2R28C mutant has unique biomolecular and biophysical characteristics distinct from the Kir4.2WT channel, which potentially elucidates its role in the pathogenesis of PD. Key Points[tpltrtarr] Inwardly rectifying potassium channels are increasingly recognized for their critical role in the complex pathogenesis of Parkinsons disease (PD) [tpltrtarr]We previously identified a genetic mutation, Kir4.2R28C, in the inwardly rectifying potassium channel Kir4.2, which strongly segregates with familial PD in a multi- generational pedigree. [tpltrtarr]This study confirms Kir4.2R28C as a loss-of-function mutation with significant dominant- negative effects, impairing channel activity even in heterozygous conditions. [tpltrtarr]The Kir4.2R28C mutation significantly reduces overall protein levels, impairs protein stability and disrupts plasma membrane trafficking in in vitro cell models.

cell biology↗

bioRxiv2022

Estimating the effectiveness of control and eradication actions on African swine fever transmission in commercial swine populations in the United States

Given the proximity of African swine fever (ASF) to the U.S., there is an urgent need to better understand the possible dissemination pathways of the virus within the U.S. swine industry and to evaluate mitigation strategies. Here, we extended PigSpread, a farm-level spatially-explicit stochastic compartmental transmission model incorporating six transmission routes including between-farm swine movements, vehicle movements, and local spread, to model the dissemination of ASF. We then examined the effectiveness of control actions similar to the ASF national response plan. The average number of secondary infections during the first 60 days of the outbreak was 49 finisher farms, 17 nursery farms, 5 sow farms, and less than one farm in other production types. The between-farm movements of swine were the predominant route of ASF transmission with an average contribution of 71.1%, while local spread and movement of vehicles were less critical with average contributions of 14.6% and 14.4%. We demonstrated that the combination of quarantine, depopulation, movement restrictions, contact tracing, and enhanced surveillance, was the most effective mitigation strategy, resulting in an average reduction of 79.0% of secondary cases by day 140 of the outbreak. Implementing these control actions led to a median of 495,619 depopulated animals, 357,789 diagnostic tests, and 54,522 movement permits. Our results suggest that the successful elimination of an ASF outbreak is likely to require the deployment of all control actions listed in the ASF national response plan for more than 140 days, as well as estimating the resources needed for depopulation, testing, and movement permits under these controls.

ecology↗

bioRxiv2020

Interrogation of SLFN11 in pediatric sarcomas uncovers an unexpected biological role and a novel therapeutic approach to overcoming resistance to replicative stress

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress, and SLFN11 gene silencing has been implicated as a common mechanism of drug resistance in tumors in adults. We found SLFN11 to be widely expressed in our cohort of pediatric sarcomas. In sarcoma cell lines, protein expression strongly correlated with response to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN), with SLFN11 knockout resulting in significant loss of sensitivity in vitro and in vivo. However, SLFN11 expression was not associated with favorable outcomes in a retrospective analysis of our patient cohort; instead, the protein was retained and promoted tumor growth and evasion. Furthermore, we show that pediatric sarcomas develop resistance to TAL and IRN through impaired intrinsic apoptosis, and that resistance can be reversed by selective inhibition of BCL-XL. Statement of SignificanceThe role of SLFN11 in pediatric sarcomas has not been thoroughly explored. In contrast to its activity in adult tumors, SLFN11 did not predict favorable outcomes in pediatric patients, was not silenced, and promoted tumor growth. Resistance to replicative stress in SLFN11-expressing sarcomas was reversed by selective inhibition of BCL-XL.

cancer biology↗