Biology subjects

Mindrebo, J. T.

Publications and source records attributed to Mindrebo, J. T..

6 recordsLinked to original sources

bioRxiv2026

Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

cell biology↗

bioRxiv2025

Cryo-EM Reveals Regulatory Mechanisms Governing Substrate Selection and Activation of Human LONP1

The human AAA+ protease LONP1 plays a central role in maintaining mitochondrial proteostasis. LONP1 processes a vast array of substrates, ranging from damaged or unfolded proteins to specific subunits stably integrated into respiratory complexes. Previous cryo-EM studies of LONP1 uncovered two distinct conformational states corresponding to inactive or active forms of the enzyme. While these states have shed light on the intricacies of LONP1 substrate translocation and proteolytic processing, little is known about the decision-making involved in LONP1 substrate engagement and subsequent initiation of its unfoldase activity. Here, we use cryo-EM to determine a novel ADP-bound, C3-symmetric intermediate state of LONP1 (LONP1C3) with putative substrate "fold-sensing" capabilities. Our biochemical and structural data indicate that LONP1C3 is an on-pathway intermediate and that is stabilized by interaction with folded substrates. Moreover, we identify additional symmetric and asymmetric conformational states, including a two-fold symmetric split-hexamer conformation, that we associate with the transition from LONP1C3 to LONP1ENZ. We propose that the C3-state regulates substrate selection and enables LONP1 to efficiently surveil the matrix proteome to ensure selective removal of damaged and dysfunctional proteins as well as privileged LONP1 substrates. These findings collectively provide further mechanistic insights into LONP1 substrate recruitment and engagement and inform on its diverse roles in maintaining homeostasis within the mitochondria.

molecular biology↗

bioRxiv2024

Structural and mechanistic studies on human LONP1 redefine the hand-over-hand translocation mechanism

AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to thread substrates through their central pore. However, the fundamental aspects of the mechanisms governing motor function and substrate processing within specific AAA+ families remain unresolved. We used cryo-electron microscopy to structurally interrogate reaction intermediates from in vitro biochemical assays to inform the underlying regulatory mechanisms of the human mitochondrial AAA+ protease, LONP1. Our results demonstrate that substrate binding allosterically regulates proteolytic activity, and that LONP1 can adopt a configuration conducive to substrate translocation even when the ATPases are bound to ADP. These results challenge the conventional understanding of the hand-over-hand translocation mechanism, giving rise to an alternative model that aligns more closely with biochemical and biophysical data on related enzymes like ClpX, ClpA, the 26S proteasome, and Lon protease.

molecular biology↗

bioRxiv2022

Mechanism-based crosslinking probes capture E. coli ketosynthase FabB in conformationally-distinct catalytic states.

Ketosynthases (KS) catalyse essential carbon-carbon bond forming reactions in fatty acid biosynthesis using a two-step, ping-pong reaction mechanism. In E. coli, there are two homodimeric elongating KSs, FabB and FabF, both of which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 14 carbons, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, we solved two crosslinked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty acid crosslinking probes that approximate catalytic steps. Both homodimeric structures possess asymmetric substrate binding pockets, suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active site gating residue, F392, potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based crosslinking methods to capture and elucidate at near atomic resolution conformational transitions accompanying KS-mediated catalysis. SynopsisCrystal structures of KS-ACP crosslinked complex elucidate chain length preference and substrate processing mechanism of E. coli FabB.

biochemistry↗

bioRxiv2021

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

biochemistry↗

bioRxiv2020

Structural and Dynamic Basis of Molecular Recognition between Acyltransferase and Carrier Protein in E. coli Fatty Acid Synthesis

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity. Significance StatementThe essential role of acyltransferases (ATs) in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways, namely the selection and loading of starter and extender units onto acyl carrier proteins (ACPs), relies on catalytically productive ACP-AT interactions. Here, we describe and interrogate the first structure of a type II FAS malonyl-CoA:ACP-transacylase (MAT) in covalent complex with its cognate ACP. We combine structural, mutational, crosslinking and kinetic data with molecular dynamics simulations to describe a highly flexible and robust protein-protein interface, substrate-induced active site reorganization, and key subdomain motions that likely govern FAS function. These findings strengthen a mechanistic understanding of molecular recognitions between ACPs and partner enzymes and provide new insights for engineering AT-dependent biosynthetic pathways.

biochemistry↗