Biology subjects

Wolfe, M. S.

Publications and source records attributed to Wolfe, M. S..

13 recordsLinked to original sources

bioRxiv2026

So similar, yet so different: α-synuclein recognition of the isoform-specific VDAC protein-lipid interface

In mammals, VDAC exists as three isoforms--VDAC1, VDAC2, and VDAC3--each characterized by a distinct tissue-dependent distribution and physiologic role. Isoform-specific knockout studies in cells and mice demonstrate distinct physiological roles for each VDAC isoform. The isoforms share about 70% sequence homology and a conserved {beta}-barrel structure and, when reconstituted into planar lipid membranes, form similar highly conductive, anion-selective, voltage-gated channels. A natural question, then, is how relatively minor differences in isoform sequences lead to striking differences in their interactions with protein partners and, consequently, in their physiological functions. To address this question, we used a small, intrinsically disordered cytosolic protein -synuclein (Syn) as a sensitive molecular probe. A comparative study with recombinant VDACs reconstituted into planar lipid membranes shows that, depending on the isoform, the Syn-induced blockage exhibits up to 100-fold differences in on-rates and equilibrium constants. The isoforms also display distinct translocation regimes for Syn, as evidenced by altered dependences of the off-rates on the applied voltage. Deletion of cysteine residues has a significant effect on Syn binding. Combined with corresponding data obtained for VDAC2 substates--which show an order of magnitude variation in on-rates while maintaining the conserved equilibrium constants--and the results of molecular dynamics simulations, these findings point to a previously unrecognized step in VDAC inhibition by Syn: an isoform-specific Syn interaction with the VDAC protein-lipid interface. This finding opens a new paradigm for understanding VDAC isoform functions and developing isoform-specific therapeutics.

biophysics↗

bioRxiv2026

Identifying Novel Estrogenic Mitochondrial Targets in Hypothalamic Proopiomelanocortin Neurons by Chemoproteomics

Loss of estrogens at menopause is linked to impaired brain metabolism and increased risk of Alzheimers disease (AD). However, estrogen replacement therapies are limited due to the deleterious effects of estrogen on peripheral organs and increased risk of vascular dementia. We have developed a non-steroidal estrogenic compound, STX, which does not bind to the classical estrogen receptors and {beta}, but mimics estrogenic signaling in the central nervous system (CNS) without the peripheral reproductive actions. STX is protective against neurodegeneration in stroke and AD models, but its molecular targets are unknown. Here, we identified and validated STX neural targets using chemoproteomic, molecular biological, electrophysiological and metabolic assays of hypothalamic proopiomelanocortin (POMC) neurons. Chemoproteomic profiling identified voltage dependent anion channels (VDAC1-3) as major intracellular binding partners in mHypo43 (POMC) cells. Based on quantitative single-cell PCR, Vdac2 was identified as the dominant isoform in female hypothalamic POMC neurons. Seahorse metabolic flux analyses showed that STX potently increased glycolysis, oxidative respiration and mitochondrial ATP production in mHypo43 cells. Nanomolar concentrations of STX enhanced VDAC2 voltage-dependent gating in reconstituted lipid membranes and shifted the low-conductance states toward anion selectivity, consistent with increased ATP flux. Together, these findings reveal a mechanism for the neuroprotective effects of STX through enhancing mitochondrial bioenergetics and modulating VDAC channel properties, potentially increasing cellular energy stores. Therefore, this work identifies previously unrecognized estrogenic mitochondrial targets and provides a mechanistic basis for the neuroprotective actions of STX relevant to menopause-associated brain vulnerability.

neuroscience↗

bioRxiv2025

AHA1 regulates Aβ production via modulation of APP expression and γ-secretase assembly

Deposition of amyloid {beta}-protein (A{beta}) is a hallmark of Alzheimers disease (AD), produced by {gamma}-secretase-mediated cleavage of amyloid precursor protein (APP). The 90-kDa heat shock protein (Hsp90) co-chaperone, activator of Hsp90 ATPase homolog 1 (AHA1), is known to promote the accumulation of toxic tau species; however, its effects on A{beta} production remain unclear. Here, we show that knockdown of endogenous AHA1 decreases A{beta} generation and reduces APP and {gamma}-secretase components, whereas AHA1 overexpression elevates A{beta} production and the expression of these proteins. The AHA1-E67K mutant, which has impaired Hsp90 binding, lowers A{beta} production and the levels of APP and {gamma}-secretase components compared with wild-type AHA1. AHA1 associates with APP and immature {gamma}-secretase components, including anterior pharynx-defective phenotype 1 (APH1), indicating its role in APP proteolysis and A{beta} production. Disruption of the AHA1/Hsp90 complex--through AHA1 knockdown, the E67K mutant, or a small-molecule inhibitor--reduces {gamma}-secretase assembly. Familial AD mutations in APP-C99 and presenilin-1 (PS1) increase AHA1, Hsp90, APP, and APH1 expression, enhancing A{beta} production. Importantly, AHA1 knockdown decreases abnormal A{beta} generation and C99, PS1-CTF, and APH1 levels in mutant APP cells, while AHA1 overexpression enhances A{beta} production in PS1 mutant cells. Collectively, these findings reveal that AHA1 regulates A{beta} production by modulating APP expression and {gamma}-secretase assembly, establishing AHA1 as a potential target for therapeutic intervention in AD.

neuroscience↗

bioRxiv2025

A C. elegans model of familial Alzheimer's disease shows age-dependent synaptic degeneration independent of amyloid β-peptide

The membrane-embedded {gamma}-secretase complex is involved in the intramembrane cleavage of [~] 150 substrates. Cleavage of amyloid precursor protein (APP)-derived substrate C99 generates 38-43-residue secreted amyloid {beta}-peptides (A{beta}), with the aggregation-prone 42-residue form (A{beta}42) particularly implicated in the pathogenesis of Alzheimers Disease (AD). However, whether A{beta}42 is the primary driver of neurodegeneration in AD remains unclear. Dominant mutations in APP or presenilin--the catalytic component of {gamma}-secretase--cause early-onset familial AD (FAD) and reduce one or more steps in the multi-step processive proteolysis of C99 to A{beta} peptides, apparently through stabilization of {gamma}-secretase enzyme-substrate (E-S) complexes. To investigate mechanisms of neurodegeneration in FAD, we developed new C. elegans models co-expressing wild-type or FAD-mutant C99 substrate and presenilin-1 (PSEN1) variants in neurons, allowing intramembrane processing of C99 to A{beta} in vivo. We demonstrate that while FAD-mutation of either C99 or PSEN1 leads to age-dependent synaptic loss, proteolytically inactive PSEN1 did not. Designed mutations that allow stable E-S complex formation without A{beta}42 or A{beta} production likewise result in synaptic degeneration. Moreover, replacement of C99 with variants of a Notch1-based substrate revealed that disrupted processing of another {gamma}-secretase substrate can similarly lead to synaptic degeneration. These results support a model in which synaptic loss can be triggered by toxic, stalled {gamma}-secretase E-S complexes in the absence of A{beta} production and not by simple loss of proteolytic function. This new C. elegans system provides a powerful platform to study the role of dysfunctional {gamma}-secretase substrate processing in FAD pathogenesis. SIGNIFICANCEDominantly inherited familial Alzheimers disease (FAD) is caused by mutations in the enzyme or substrate that produces amyloid {beta}-peptides (A{beta}). These mutations alter enzyme processing of substrate to A{beta} and can skew production of A{beta} to aggregation-prone forms that deposit as pathological plaques. Nevertheless, whether A{beta} is the primary driver of AD remains unresolved. Recent evidence supports a model in which loss of neuronal connections in FAD is due to the stalled enzymatic process of A{beta} production, rather than the A{beta} products. Using the roundworm C. elegans as a genetic model system, we show here that the stalled process itself, rather than A{beta} or reduced enzyme activity, can trigger loss of neuronal connections with age in this simple model animal.

neuroscience↗

bioRxiv2025

γ-Secretase-mediated endoproteolysis of neuregulin-1 and E-cadherin

{gamma}-Secretase is an intramembrane protease complex, with nearly 150 substrates that are cleaved within their transmembrane domains (TMD). Amyloid Precursor Protein (APP) is the most widely studied, as processive proteolysis by {gamma}-secretase releases the amyloid-{beta}-peptide (A{beta}) implicated in the pathogenesis of Alzheimers disease. In contrast, proteolysis of other substrates has been little explored. The only known sequence specificity rule for {gamma}-secretase cleavage is for APP, in which phenylalanine is not tolerated at P2 with respect to any step in processive proteolysis. Recently, we found this specificity rule applies to the initial cleavage of Notch1 substrate as well. In this study, we examined the site of initial cleavage by {gamma}-secretase and explored the phenylalanine rule for two other {gamma}-secretase substrates: neuregulin1 (NRG1) and E-cadherin (CDH1). Upon incubation of recombinant substrates with purified protease complex, followed by mass spectroscopy (MS) and immunoblot analysis, initial cleavage products for NRG1 and CDH1 were identified. Two cleavage sites were observed in the NRG1 TMD, one of which matched that seen previously. However, the observed single CDH1 TMD cleavage site differed from the reported cytosolic cleavage site. Phenylalanine mutants of NRG1 and CDH1 in the P2 position relative to the first {gamma}-secretase cleavage site showed a shift in the cleavage site, along with reduction in total C-terminal and N-terminal products, compared to that seen with wild-type substrates. Taking together, these findings clarify the initial cleavage sites of NRG1 and CDH1 and support the intolerance of Phe at P2 position as a general rule for {gamma}-secretase substrates. For Table of Contents use only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/649652v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1cfdda7org.highwire.dtl.DTLVardef@1e10813org.highwire.dtl.DTLVardef@d94ba6org.highwire.dtl.DTLVardef@1f58c08_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Effects of transmembrane phenylalanine residues on γ-secretase-mediated Notch-1 proteolysis

{gamma}-Secretase is a presenilin-containing intramembrane aspartyl protease complex that cleaves within the transmembrane domain (TMD) of nearly 150 substrates, with the amyloid precursor protein (APP) being the most well studied. APP cleavage by {gamma}-secretase generates amyloid {beta}-peptides (A{beta}) that pathologically deposit in Alzheimers disease. APP TMD substrate undergoes initial endoproteolysis ({varepsilon}-cleavage) followed by processive carboxypeptidase trimming of long A{beta} intermediates in [~]tripeptide intervals. Although {gamma}-secretase cleavage of Notch1 is essential in developmental biology and altered in many cancers, the processing of this cell-surface receptor is relatively understudied. Only one sequence specificity rule is known for {gamma}-secretase substrate processing: Aromatic residues such as phenylalanine are not tolerated in the P2 position with respect to any processing event on the APP TMD. Here we show using biochemical and mass spectrometry (MS) techniques that this specificity rule holds for Notch1 as well. Analysis of products from the reactions of purified enzyme complex and Notch1 TMD substrate variants revealed that P2 Phe relative to {varepsilon}-site cleavage reduced proteolysis and shifted initial cleavage N-terminally by one residue. Double Phe mutation near the {varepsilon} site resulted in reduced proteolysis with shifting to two major initial cleavage sites, one N-terminally and one C-terminally, both of which avoid Phe in the P2 position. Additionally, three natural Phe residues were mutated to corresponding residues in the APP TMD, which led to increased {varepsilon} proteolysis. Thus, Phe residues can affect the enzyme reaction rate as well as cleavage site specificity in the Notch1 TMD.

biochemistry↗

bioRxiv2024

Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

Missense mutations in the amyloid precursor protein (APP) and presenilin-1 (PSEN1) cause early-onset familial Alzheimers disease (FAD) and alter proteolytic production of secreted 38-to-43-residue amyloid {beta}-peptides (A{beta}) by the PSEN1-containing {gamma}-secretase complex, ostensibly supporting the amyloid hypothesis of pathogenesis. However, proteolysis of APP substrate by {gamma}-secretase is processive, involving initial endoproteolysis to produce long A{beta} peptides of 48 or 49 residues followed by carboxypeptidase trimming in mostly tripeptide increments. We recently reported evidence that FAD mutations in APP and PSEN1 cause deficiencies in early steps in processive proteolysis of APP substrate C99 and that this results from stalled {gamma}-secretase enzyme-substrate and/or enzyme-intermediate complexes. These stalled complexes triggered synaptic degeneration in a C. elegans model of FAD independently of A{beta} production. Here we conducted full quantitative analysis of all proteolytic events on APP substrate by {gamma}-secretase with six additional PSEN1 FAD mutations and found that all six are deficient in multiple processing steps. However, only one of these (F386S) was deficient in certain trimming steps but not in endoproteolysis. Fluorescence lifetime imaging microscopy in intact cells revealed that all six PSEN1 FAD mutations lead to stalled {gamma}-secretase enzyme-substrate/intermediate complexes. The F386S mutation, however, does so only in A{beta}-rich regions of the cells, not in C99-rich regions, consistent with the deficiencies of this mutant enzyme only in trimming of A{beta} intermediates. These findings provide further evidence that FAD mutations lead to stalled and stabilized {gamma}-secretase enzyme-substrate and/or enzyme-intermediate complexes and are consistent with the stalled process rather than the products of {gamma}-secretase proteolysis as the pathogenic trigger.

biochemistry↗

bioRxiv2023

Molecular Dynamics Activation of γ-Secretase for Cleavage of Notch1 Substrate

{gamma}-Secretase is an intramembrane aspartyl protease complex which cleaves the transmembrane domain of over 150 peptide substrates, including amyloid precursor protein (APP) and the Notch family of receptors, via two conserved aspartates D257 and D385 in the Presenilin-1 (PS1) catalytic subunit. However, while the activation of {gamma}-secretase for cleavage of APP has been widely studied, the cleavage of Notch by {gamma}-secretase remains poorly explored. Here, we combined Gaussian accelerated Molecular Dynamics (GaMD) simulations and mass spectrometry (MS) analysis of proteolytic products to present the first dynamic models for cleavage of Notch by {gamma}-secretase. MS showed that {gamma}-secretase cleaved the WT Notch at Notch residue G34, while cleavage of L36F mutant Notch occurred at Notch residue C33. Initially, we prepared our simulation systems starting from the cryoEM structure of Notch-bound {gamma}-secretase (PDB: 6IDF) and failed to capture the proper cleavages of WT and L36F Notch by {gamma}-secretase. We then discovered an incorrect registry of the Notch substrate in the PS1 active through alignment of the experimental structure of Notch-bound (PDB: 6IDF) and APP-bound {gamma}-secretase (PDB: 6IYC). Every residue of APP substrate was systematically mutated to the corresponding Notch residue to prepare a resolved model of Notch-bound {gamma}-secretase complexes. GaMD simulations of the resolved model successfully captured {gamma}-secretase activation for proper cleavages of both WT and L36F mutant Notch. Our findings here provided mechanistic insights into the structural dynamics and enzyme-substrate interactions required for {gamma}-secretase activation for cleavage of Notch and other substrates.

biophysics↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗

bioRxiv2021

Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid β-peptide production

A series of analogs based on a prototype aryl aminothiazole {gamma}-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid {beta}-peptides (A{beta}), generated through processive proteolysis of precursor protein substrate by {gamma}-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of A{beta} production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in A{beta}37 and A{beta}38 production without parallel decreases in their precursors A{beta}40 and A{beta}42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of A{beta} peptides [≥]43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by {gamma}-secretase. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2021

Mechanism of Tripeptide Trimming by γ-Secretase

The membrane-embedded {gamma}-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid {beta}-peptides (A{beta}) of Alzheimers disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by {gamma}-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (A{beta}49[->]A{beta}46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was similar for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of A{beta}49 by {gamma}-secretase. The starting structure was active {gamma}-secretase bound to A{beta}49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of {gamma}-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of A{beta}49 to A{beta}46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and A{beta}49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by {gamma}-secretase. Significance statementProduction of amyloid {beta}-peptide (A{beta}) of Alzheimers disease (AD) from its precursor protein requires a series of proteolytic events carried out by the membrane-embedded {gamma}-secretase complex. Mutations in the substrate and enzyme that produce A{beta} cause hereditary AD and these mutations affect tripeptide trimming of initially formed long A{beta} peptides by {gamma}-secretase. Little is known about the structural mechanism of this trimming process. Here, we have developed a molecular dynamic model for the first step of trimming (A{beta}49 to A{beta}46) by {gamma}-secretase. Conformational changes in the enzyme-substrate complex to set up this trimming step require the presence of N-terminally charged endoproteolytic cleavage co-product. Computational effects of AD-causing mutations in A{beta}49 on the trimming process were validated through biochemical experiments.

biochemistry↗

bioRxiv2021

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimers disease is processively cut by {gamma}-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis--composed of a helical peptide inhibitor linked to a transition-state analog--that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both exosite and active site as demonstrated by inhibitor cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with {gamma}-secretase, ultimately aiding rational drug design.

biochemistry↗

bioRxiv2020

Mechanisms of γ-Secretase Activation and Substrate Processing

Amyloid {beta}-peptide, the principal component of characteristic cerebral plaques of Alzheimers disease (AD), is produced through intramembrane proteolysis of the amyloid precursor protein (APP) by {gamma}-secretase. Despite the importance in pathogenesis of AD, the mechanisms of intramembrane proteolysis and substrate processing by {gamma}-secretase remain poorly understood. Here, complementary all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method and biochemical experiments were combined to investigate substrate processing of wildtype and mutant APP by {gamma}-secretase. The GaMD simulations captured spontaneous activation of {gamma}-secretase, with hydrogen bonded catalytic aspartates and water poised for proteolysis of APP at the {varepsilon} cleavage site. Furthermore, GaMD simulations revealed that familial AD mutations I45F and T48P enhanced the initial {varepsilon} cleavage between residues Leu49-Val50, while M51F mutation shifted the {varepsilon} cleavage site to the amide bond between Thr48-Leu49. Detailed analysis of the GaMD simulations allowed us to identify distinct low-energy conformational states of {gamma}-secretase, different secondary structures of the wildtype and mutant APP substrate, and important active-site sub-pockets for catalytic function of the enzyme. The simulation findings were highly consistent with experimental analyses of APP proteolytic products using mass spectrometry and western blotting. Taken together, the GaMD simulations and biochemical experiments have enabled us to elucidate the mechanisms of {gamma}-secretase activation and substrate processing, which should facilitate rational computer-aided drug design targeting this functionally important enzyme.

biochemistry↗