Biology subjects

Kemp, G.

Publications and source records attributed to Kemp, G..

6 recordsLinked to original sources

bioRxiv2026

The modulatory effects of Hericium erinaceus and donepezil on amyloid accumulation and autophagy-associated clearance in Aβ1-42-induced N2a cells

Background and objective: Alzheimers disease involves imbalanced amyloid-{beta} (A {beta}) production and clearance, supporting investigation of adjuncts to symptomatic treatment. This study evaluated whether methanolic Hericium erinaceus extract could support recovery after A{beta}1-42-induced injury, alone and with donepezil, through modulation of {beta}-secretase (BACE1) activity or autophagy-associated clearance. Methods: Extract composition and BACE1 inhibition were assessed using liquid chromatography-mass spectrometry (LC-MS) and a fluorescence resonance energy transfer assay, respectively. Neuro-2a cells exposed to A{beta}1-42 (2.42 M) for 6 hours received individual or combined treatments for 24 hours. Cell viability was assessed using WST-1, combination interactions using Bliss independence and protein responses using Western blotting with paired bafilomycin A1 conditions across three independent experiments. Results: LC-MS identified ergothioneine and trace hericenone D. The extract produced an apparent concentration-dependent BACE1-inhibitory response (IC50, 69.97 {+/-} 36.10 g/mL) and significantly increased post-injury viability at 40g/mL (150.2 {+/-} 12.11% of the A {beta} induced control; p = 0.0070). Although the full combination matrix was predominantly additive (Bliss score, 1.95), 51.1 M donepezil with 24.9 g/mL extract produced local synergy (score, 34.29) and the highest observed viability. This pair showed the most coordinated directional protein pattern, with lower mean levels of amyloid precursor protein, A{beta}, and BACE1, and p62/A {beta} accumulation following lysosomal blockade. Western blot differences were not statistically significant. Conclusion: These integrated findings support H. erinaceus as a candidate adjunct to donepezil, with possible complementary effects on amyloidogenic processing and intracellular clearance. Further validation of autophagic flux is needed to confirm the proposed dual response. Keywords: Hericium erinaceus; donepezil; BACE1; amyloid- {beta} ; autophagy; combination treatment

neuroscience↗

bioRxiv2026

Buried Ion-Pairs Reshape Folding Pathways of Artificial Proteins

Ion-pairs are central charged motifs that control the biological function of several proteins. Yet, the physical principles linked to the formation of ion-pairs during protein folding remain poorly understood. Here, we show that buried ion-pairs of de novo-designed helical bundle proteins reshape the folding pathways by introducing kinetic barriers that are overcome by a hydrophobic collapse linked to packing of the protein core. By combining protein design with co-translational folding studies, nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics simulations, we show that introduction of a buried ion-pair leads to a shifted folding transition that balances desolvation and electrostatic interactions within the protein and the bulk solvent. Moreover, we find that the folding pathway is sensitive to modifications in non-polar interactions and motifs that electrostatically shield the ion-pair from its surroundings. Our combined findings provide a blueprint for understanding how functional motifs affect protein folding and how mutation of charged networks alter the stability of proteins.

biophysics↗

bioRxiv2025

Arachidonic acid modulates fluconazole-induced responses in Candida albicans biofilms

Candida albicans is a human commensal, which causes opportunistic infections with high morbidity and mortality and is characterised by the production of resistant biofilms. Polyunsaturated fatty acids, such as arachidonic acid, can increase azole susceptibility of C. albicans biofilms significantly. However, the underlining mechanism is not known. We investigated the effect of arachidonic acid on known fluconazole resistance mechanisms namely, overexpression of ERG11, increased ergosterol content, oxidative stress resistance, as well as expression and activity of the efflux pump, Cdr1p. Upregulation of ERG11 was observed in biofilms exposed to fluconazole. However, this was reversed in the presence of arachidonic acid, even in the presence of fluconazole. Furthermore, arachidonic acid downregulated the fluconazole-induced oxidative stress response of C. albicans. Previous transcriptome data indicated a significant increase in CDR1 expression during early biofilm formation in the presence of arachidonic acid. However, we found that efflux activity was reduced in the presence of arachidonic acid, which indicates the loss of function of the membrane-associated protein. This contradictory phenomenon was further investigated by determining the effect of arachidonic acid on the localisation and phosphorylation of Cdr1p (ProteomeXchange identifier PXD070958). Our results show that the presence of arachidonic acid may cause mislocalisation and changes in the phosphorylation of Cdr1p; all of which could impact its activity. These results demonstrate that multiple mechanisms are potentially involved in the arachidonic acid-induced increased fluconazole susceptibility and allows for further exploration of lipid-mediated modulation of antifungal responses.

microbiology↗

bioRxiv2020

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing E. coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol:disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Cotranslational folding cooperativity of contiguous domains of α-spectrin

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein -spectrin using Force Profile Analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each others folding in distinct ways that may be important for the efficient assembly of -spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in pN. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of [~]15 pN during cotranslational folding. SignificanceIn living cells, proteins are produced in a sequential way by ribosomes. This vectoral process allows the growing protein chain to start to fold before translation has been completed. Thereby, cotranslational protein folding can be significantly different than the folding of a full-length protein in isolation. Here we show how structurally similar repeat domains, normally produced as parts of a single long polypeptide, affect the cotranslational folding of their neighbors. This provides insight into how the cell may efficiently produce multidomain proteins, paving the way for future studies in vivo or with chaperones. We also provide an estimated magnitude of the mechanical force on the nascent chain generated by cotranslational folding, calculated from biochemical measurements and molecular dynamics simulations.

biochemistry↗

bioRxiv2018

Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

We have characterized the cotranslational folding of two small protein domains of different folds - the a-helical N-terminal domain of HemK and the {beta}-rich FLN5 filamin domain - by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (Force-Profile Analysis - FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, PET, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.

molecular biology↗