scholarly journals Mechanism and ultrasensitivity in Hedgehog signaling revealed by Patched1 disease mutations

2021 ◽  
Vol 118 (6) ◽  
pp. e2006800118
Author(s):  
Kostadin Petrov ◽  
Taciani de Almeida Magalhaes ◽  
Adrian Salic
Keyword(s):  
Mathematical Modeling ◽  
Catalytic Activity ◽  
Single Cell ◽  
Birth Defects ◽  
Hedgehog Signaling ◽  
Loss Of Function ◽  
Functional Assays ◽  
Rich Domain ◽  
Disease Mutations ◽  
Cysteine Rich Domain

Hedgehog signaling is fundamental in animal embryogenesis, and its dysregulation causes cancer and birth defects. The pathway is triggered when the Hedgehog ligand inhibits the Patched1 membrane receptor, relieving repression that Patched1 exerts on the GPCR-like protein Smoothened. While it is clear how loss-of-function Patched1 mutations cause hyperactive Hedgehog signaling and cancer, how other Patched1 mutations inhibit signaling remains unknown. Here, we develop quantitative single-cell functional assays for Patched1, which, together with mathematical modeling, indicate that Patched1 inhibits Smoothened enzymatically, operating in an ultrasensitive regime. Based on this analysis, we propose that Patched1 functions in cilia, catalyzing Smoothened deactivation by removing cholesterol bound to its extracellular, cysteine-rich domain. Patched1 mutants associated with holoprosencephaly dampen signaling by three mechanisms: reduced affinity for Hedgehog ligand, elevated catalytic activity, or elevated affinity for the Smoothened substrate. Our results clarify the enigmatic mechanism of Patched1 and explain how Patched1 mutations lead to birth defects.

scholarly journals Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain

2019 ◽  
Vol 5 (7) ◽  
pp. eaau4202 ◽  
Author(s):  
Janna M. Bigalke ◽  
Shintaro Aibara ◽  
Robert Roth ◽  
Göran Dahl ◽  
Euan Gordon ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Neurological Diseases ◽  
Receptor Activation ◽  
Loss Of Function ◽  
Calcium Binding Site ◽  
Signaling Complex ◽  
Rich Domain ◽  
Extracellular Region ◽  
Domain 3 ◽  
Cysteine Rich Domain

Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFRα2 and determined its structure at 5.7-Å resolution by cryo-EM. The proteins form an assembly through RET-GFRα2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFRα2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.

Zinc co-ordination by the DHHC cysteine-rich domain of the palmitoyltransferase Swf1

2013 ◽  
Vol 454 (3) ◽  
pp. 427-435 ◽  
Author(s):  
Ayelén González Montoro ◽  
Rodrigo Quiroga ◽  
Javier Valdez Taubas
Keyword(s):  
Amino Acids ◽  
Tertiary Structure ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Essential Amino Acids ◽  
Zinc Binding ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Rich Domain ◽  
Cysteine Rich Domain

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with fatty acids. This modification is catalysed by a family of PATs (palmitoyltransferases), characterized by the presence of a 50-residue long DHHC-CRD (Asp-His-His-Cys cysteine-rich domain). To gain knowledge on the structure–function relationships of these proteins, we carried out a random-mutagenesis assay designed to uncover essential amino acids in Swf1, the yeast PAT responsible for the palmitoylation of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modelling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, co-ordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the co-ordination of zinc were found to be essential for Swf1 function in the screen. Moreover, these mutations result in unstable proteins, in agreement with a structural role for these zinc fingers. The conservation of amino acids predicted to form each zinc-binding pocket suggests a shared function, as the selective pressure to maintain them is lost upon mutation of one of them. A Swf1 orthologue that lacks one of the zinc-binding pockets is able to complement a yeast swf1∆ strain, possibly because a similar fold can be stabilized by hydrogen bonds instead of zinc co-ordination. Finally, we show directly that recombinant Swf1 DHHC-CRD is able to bind zinc. Sequence analyses of DHHC domains allowed us to present models of the zinc-binding properties for all PATs.

scholarly journals Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Giovanni Luchetti ◽  
Ria Sircar ◽  
Jennifer H Kong ◽  
Sigrid Nachtergaele ◽  
Andreas Sagner ◽  
...  
Keyword(s):  
Structural Analysis ◽  
G Protein ◽  
Hedgehog Signaling ◽  
Cell Communication ◽  
G Protein Coupled Receptors ◽  
Rich Domain ◽  
Hh Signaling ◽  
G Protein Coupled ◽  
Orphan Gpcr ◽  
Cysteine Rich Domain

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.

scholarly journals Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Sigrid Nachtergaele ◽  
Daniel M Whalen ◽  
Laurel K Mydock ◽  
Zhonghua Zhao ◽  
Tomas Malinauskas ◽  
...  
Keyword(s):  
Small Molecules ◽  
Drug Target ◽  
Hedgehog Signaling ◽  
Rich Domain ◽  
Regulatory Module ◽  
Wnt Ligands ◽  
Binding Groove ◽  
And Function ◽  
Endogenous Lipids ◽  
Cysteine Rich Domain

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants.

scholarly journals RASopathy Mutations Demonstrate the Critical Function of the Cysteine-rich Domain in Maintaining BRAF in an Autoinhibited State

2021 ◽  
Author(s):  
Russell Spencer-Smith ◽  
Elizabeth M Terrell ◽  
Christine Insinna ◽  
Constance Agamasu ◽  
Morgan E Wagner ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
High Frequency ◽  
Human Cancer ◽  
Critical Role ◽  
Critical Function ◽  
Disease States ◽  
Rich Domain ◽  
Predominant Factor ◽  
Cysteine Rich Domain

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD is known for roles in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, how these differing activities impact BRAF function in normal and disease states is not well-characterized. Here, we analyze a panel of BRAF-CRD mutations and find that they can be classified into three groups based on their ability to relieve autoinhibition and/or enhance PS binding, with relief of autoinhibition being the predominant factor determining mutation severity. Comparison of the BRAF and CRAF CRDs further reveals that the BRAF-CRD is a stronger mediator of both autoinhibition and PS binding. Moreover, given the increased catalytic activity of BRAF versus CRAF, our findings indicate a more critical role for CRD-mediated autoinhibition in BRAF regulation, consistent with the high frequency of mutations that disrupt this function in the RASopathies.

scholarly journals The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors

2015 ◽  
Vol 6 ◽  
Author(s):  
Nicholas V. L. Yap ◽  
Fiona J. Whelan ◽  
Dawn M. E. Bowdish ◽  
G. Brian Golding
Keyword(s):  
Scavenger Receptor ◽  
Scavenger Receptors ◽  
Class A ◽  
Rich Domain ◽  
Cysteine Rich Domain

scholarly journals ATRT-13. DIFFERENT CELLS OF ORIGIN PAVE THE WAY FOR MOLECULAR HETEROGENEITY IN RHABDOID TUMORS

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii278-iii278
Author(s):  
Monika Graf ◽  
Marta Interlandi ◽  
Natalia Moreno ◽  
Dörthe Holdhof ◽  
Viktoria Melcher ◽  
...  
Keyword(s):  
Single Cell ◽  
Expression Patterns ◽  
Time Frame ◽  
Precursor Cells ◽  
Genetically Engineered ◽  
Molecular Heterogeneity ◽  
Loss Of Function ◽  
Cellular Origin ◽  
Cells Of Origin ◽  
Rhabdoid Tumors

Abstract Rhabdoid tumors (RT) are rare but highly aggressive pediatric neoplasms. These tumors carry homozygous loss-of-function alterations of SMARCB1 in almost all cases with an otherwise low mutational load. RT arise at different intracranial (ATRT) as well as extracranial (MRT) anatomical sites. Three main molecular subgroups (ATRT-SHH, ATRT-TYR, ATRT-MYC) have been characterized for ATRT which are epigenetically and clinically diverse, while MRT show remarkable similarities with ATRT-MYC distinct from ATRT-SHH and ATRT-TYR. Even though there are hypotheses about various cells of origin among RT subgroups, precursor cells of RT have not yet been identified. Previous studies on the temporal control of SMARCB1 knockout in genetically engineered mouse models have unveiled a tight vulnerable time frame during embryogenesis with regard to the susceptibility of precursor cells to result in RT. In this study, we employed single-cell RNA sequencing to describe the intra- and intertumoral heterogeneity of murine ATRT-SHH and -MYC as well as extracranial MYC tumor cells. We defined subgroup-specific tumor markers for all RT classes but also observed a notable overlap of gene expression patterns in all MYC subgroups. By comparing these single-cell transcriptomes with available single-cell maps of early embryogenesis, we gained first insights into the cellular origin of RT. Finally, unsupervised clustering of published human RT methylation data and healthy control tissues confirmed the existence of different cells of origin for intracranial SHH tumors and MYC tumors independent of their anatomical localizations.

scholarly journals The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

Mobile DNA ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marc Guérineau ◽  
Luiza Bessa ◽  
Séverine Moriau ◽  
Ewen Lescop ◽  
François Bontems ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Dna Cleavage ◽  
Trichoplusia Ni ◽  
Catalytic Domain ◽  
Structural Diversity ◽  
Paramecium Tetraurelia ◽  
Lysine Methylation ◽  
Unusual Structure ◽  
Rich Domain ◽  
Cysteine Rich Domain

Abstract Background Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. Results We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. Conclusions The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

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