Maia Kinnebrew
Postdoctoral Scholar, Biochemistry
https://orcid.org/0000-0002-7344-8231All Publications
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Multiple modes of cholesterol translocation in the human Smoothened receptor.
bioRxiv : the preprint server for biology
2025
Abstract
Smoothened (SMO), a member of the G Protein-Coupled Receptor superfamily, mediates Hedgehog signaling and is linked to cancer and birth defects. SMO responds to accessible cholesterol in the ciliary membrane, translocating it via a longitudinal tunnel to its extracellular domain. Reaching a complete mechanistic understanding of the cholesterol translocation process would help in the development of cancer therapies. Experimental data suggests two modes of translocation to support entry of cholesterol from outer and inner membrane leaflets, but the exact mechanism of translocation remains unclear. Using atomistic molecular dynamics simulations (~2 millisecond simulations) and biochemical assays of SMO mutants, we assess the energetic feasibilities of the two modes. We show that the highest energetic barrier for cholesterol translocation from the outer leaflet is lower than that from the inner leaflet. Mutagenesis experiments and complementary simulations of SMO mutants validate the role of critical amino acid residues along the translocation pathways. Our data suggests that cholesterol can take either pathway to enter SMO, thus explaining experimental observations in literature. Thus, our results illuminate the energetics and provide a first molecular description of cholesterol translocation in SMO.
View details for DOI 10.1101/2024.11.25.625241
View details for PubMedID 39651171
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Direct ionic stress sensing and mitigation by the transcription factor NFAT5.
Science advances
2025; 11 (8): eadu3194
Abstract
Rising temperatures and water scarcity caused by climate change are increasingly exposing our cells and tissues to ionic stress, a consequence of elevated cytoplasmic ionic strength that can disrupt protein, organelle, and genome function. Here, we unveil a single-protein mechanism for ionic strength sensing and mitigation in animal cells, one that is notably different from the analogous high osmolarity glycerol kinase cascade in yeast. The Rel family transcription factor NFAT5 directly senses intracellular ionic strength using a C-terminal prion-like domain (PLD). In response to elevated intracellular ionic strength, this PLD is necessary and sufficient to coordinate an adaptive gene expression program by recruiting the transcriptional coactivator BRD4. The purified NFAT5 PLD forms condensates in response to elevated solution ionic strength in vitro, and human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic stress in yeast. We propose that ion-sensitive conformational changes in a PLD directly regulate transcription to maintain ionic strength homeostasis in animal cells.
View details for DOI 10.1126/sciadv.adu3194
View details for PubMedID 39970224
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A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells.
Proceedings of the National Academy of Sciences of the United States of America
2024; 121 (7): e2318024121
Abstract
Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.
View details for DOI 10.1073/pnas.2318024121
View details for PubMedID 38330014
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Direct ionic stress sensing and mitigation by the transcription factor NFAT5.
bioRxiv : the preprint server for biology
2023
Abstract
Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.
View details for DOI 10.1101/2023.09.23.559074
View details for PubMedID 37886503
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The energetics and ion coupling of cholesterol transport through Patched1.
Science advances
2023; 9 (34): eadh1609
Abstract
Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between "inward-open" and solvent "occluded" states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.
View details for DOI 10.1126/sciadv.adh1609
View details for PubMedID 37611095
View details for PubMedCentralID PMC10446486
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The Energetics and Ion Coupling of Cholesterol Transport Through Patched1.
bioRxiv : the preprint server for biology
2023
Abstract
Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol -1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) we find that transition between 'inward-open' and solvent 'occluded' states is accompanied by Na + induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion-coupling stoichiometries of PTCH1 transport mechanisms, whereby 1-3 Na + or 2-3 K + couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.
View details for DOI 10.1101/2023.02.14.528445
View details for PubMedID 36824746
View details for PubMedCentralID PMC9949057
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Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain.
Science advances
2022; 8 (22): eabm5563
Abstract
Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.
View details for DOI 10.1126/sciadv.abm5563
View details for PubMedID 35658032
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Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling.
Methods in molecular biology (Clifton, N.J.)
2022; 2374: 73-87
Abstract
Cholesterol is an abundant lipid in mammalian plasma membranes that regulates the reception of the Hedgehog (Hh) signal in target cells. In vertebrates, cell-surface organelles called primary cilia function as compartments for the propagation of Hh signals. Recent structural, biochemical, and cell-biological studies have led to the model that Patched-1 (PTCH1), the receptor for Hh ligands, uses its transporter-like activity to lower cholesterol accessibility in the membrane surrounding primary cilia. Cholesterol restriction at cilia may represent the long-sought-after mechanism by which PTCH1 inhibits Smoothened (SMO), a cholesterol-responsive transmembrane protein of the G protein-coupled receptor superfamily that transmits the Hh signal across the membrane.Protein probes based on microbial cholesterol-binding proteins revealed that PTCH1 controls only a subset of the total cholesterol molecules, a biochemically defined fraction called accessible cholesterol. The accessible cholesterol pool coexists (and exchanges) with a pool of sequestered cholesterol, which is bound to phospholipids like sphingomyelin. In this chapter, we describe how to measure the accessible and sequestered cholesterol pools in live cells with protein-based probes. We discuss how to purify and fluorescently label these probes for use in flow cytometry and microscopy-based measurements of the cholesterol pools. Additionally, we describe how to modulate accessible cholesterol levels to determine if this pool regulates Hh signaling (or any other cellular process of interest).
View details for DOI 10.1007/978-1-0716-1701-4_7
View details for PubMedID 34562244
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Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes
ELIFE
2021; 10
View details for DOI 10.7554/eLife.70504.sa2
View details for Web of Science ID 000729482600001
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Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes.
eLife
2021; 10
Abstract
A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. To directly test the effect of PTCH1 on accessible cholesterol, we measured the transport activity of PTCH1 using an imaging-based assay to follow the kinetics of cholesterol extraction from the plasma membrane of live cells by methyl-beta-cyclodextrin. PTCH1 depletes accessible cholesterol in the outer leaflet of the membrane in a manner regulated by its ligand Sonic Hedgehog and the transmembrane potassium gradient. We propose that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export. Our results show that proteins can change accessible cholesterol levels in membranes to regulate signaling reaction.
View details for DOI 10.7554/eLife.70504
View details for PubMedID 34698632
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Publisher Correction: Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.
Nature genetics
2021
View details for DOI 10.1038/s41588-021-00849-4
View details for PubMedID 33762754
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Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.
Nature genetics
2021
Abstract
Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.
View details for DOI 10.1038/s41588-021-00804-3
View details for PubMedID 33731941
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Structures of vertebrate Patched and Smoothened reveal intimate links between cholesterol and Hedgehog signalling.
Current opinion in structural biology
2019; 57: 204–14
Abstract
The Hedgehog (HH) signalling pathway is a cell-cell communication system that controls the patterning of multiple tissues during embryogenesis in metazoans. In adults, HH signals regulate tissue stem cells and regenerative responses. Abnormal signalling can cause birth defects and cancer. The HH signal is received on target cells by Patched (PTCH1), the receptor for HH ligands, and then transmitted across the plasma membrane by Smoothened (SMO). Recent structural and biochemical studies have pointed to a sterol lipid, likely cholesterol itself, as the elusive second messenger that communicates the HH signal between PTCH1 and SMO, thus linking ligand reception to transmembrane signalling.
View details for DOI 10.1016/j.sbi.2019.05.015
View details for PubMedID 31247512
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Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling.
eLife
2019; 8
Abstract
Previously we proposed that transmission of the Hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.
View details for DOI 10.7554/eLife.50051
View details for PubMedID 31657721
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The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.
Nature chemical biology
2019
Abstract
Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.
View details for DOI 10.1038/s41589-019-0370-y
View details for PubMedID 31548691
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CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog.
Developmental cell
2018; 44 (1): 113–29.e8
Abstract
To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells.
View details for PubMedID 29290584
View details for PubMedCentralID PMC5792066