Table 1.

Functional involvement of Hh signaling regulatory proteins in Hh pathway and their association with cholesterol

Hh signaling proteins	Functional contribution
	Involvement in Hh signaling	Association with cholesterol
Hedgehog (Hh)	Ligands of Hh signaling serves as a morphogen, mitogen, survival factor, and guidance [11, 47, 53, 54] Hh family comprises three Hh-related genes sonic hedgehog (Shh), desert hedgehog (Dhh), and Indian hedgehog (Ihh) [86]	Various roles of cholesterol in Hh protein biogenesis and signal transmission [155] During Hh auto-processing, the N-terminal half of Hh is covalently anchored with cholesterol [61, 160]
		Cholesterol-modification of Hh is required for Hh activity and native signal transduction [62, 156]
Smoothened (Smo)	Smo encodes a serpentine protein, a member of the G-protein coupled receptor (GPCR) superfamily that is implicated as a core receptor of Hh signaling [88, 90] Smo activation involves Smo translocation from intracellular vesicle to the cell surface and its post-translational modification [91, 96] Hh-mediated Smo activity is regulated by inducing a conformational switch [94] The long C-terminal domain (C-tail) of Smo is indispensable for Hh-dependent signal transduction [95]	Cholesterol serves as an endogenous ligand for Smo [40, 154] Cholesterol binding is required for allosteric Smo activation [40, 154] Cholesterol modification of Smo is needed to reinforce and sustain the Hh signaling [41]
		Smo activity is regulated by cholesterol concentration or accessibility in the ciliary and plasma membrane [32, 40, 192]
		Local concentration of cholesterol affects the trafficking of Smo [32]
Patched 1 (Ptch1)	A twelve-span transmembrane receptor of Hh signaling [73]	Cation-powered Ptch1transporter uses distinct cation gradients power for cholesterol transport [46] Ptch1 is implicated in cholesterol efflux [187] Ptch1 modulates the intracellular cholesterol concentration [187]
	Ptch1 involves in the suppression of the Smo enrichment in the plasma membrane [87] The sterol-sensing domain of Ptch1 appears to control Smo activity through vesicular trafficking [45]
	Extracellular domains of Ptch1 bind to Hh ligand [127] Ptch1 acts as a dependence receptor [135]: Intracellular C-terminal domain modulates Ptch1 activity and has pro-apoptotic activity [134, 135] Acts as tumor-suppressor gene and restricts cell cycle progression [136, 138]	Local concentration of cholesterol affects the trafficking of Ptch1 Ptch1 regulates the Smo activation by transporting cholesterol from cells [24]
		Ptch1 acts as a lipoprotein receptor and modulates intracellular lipid homeostasis in a Hh-independent manner [185]
Dispatched (Disp)	Twelve-transmembrane membrane receptor[44] In signal-producing cells, Disp controls the release of fully functional Hh protein [37, 177]	Disp is a cation-powered transporter that requires cholesterol for Hh signal transfer [46, 177] Hh binds physically to Disp through cholesterol moiety [164] Disp facilitates efficient secretion of the cholesterol modified Hh ligand [44, 164] Disp regulates long-range cholesterol modified Hh signal transmission [163]
Cell-adhesion-molecule related/down-regulated by oncogene (CDON) Bioregional Cdon-binding protein (BOC)	CDON and BOC co-receptors recruit Scube-Hh into the responding cells as a ternary complex with CDON/BOC [38]
Growth-arrest-specific (Gas1)	Gas1 co-receptor catalyzes Hh transfer to Ptch receptor [170] Gas 1 acts as a positive regulator of the Hh signaling [172] Gas1 extends the range of Hh action by assisting its signaling [171]	Gas1 interacts with Hh in a lipid-dependent manner and dissociates its interaction with Scube [39]
Suppressor of fused (Sufu)	Sufu, a scaffold protein, acts as a negative regulator of the Hh signaling acting on Gli [120, 121]
Signal peptide-CUB-EGF domain-containing protein (Scube)	Scube is secreted glycoprotein, and Scube family of proteins are Scube1, Scube2, Scube3, and its activity is required for Hh signal transduction [70, 162] Scube activity is essential for efficient Hh secretion and long-range Hh signal transmission [71, 162, 164] Scube acts as a chaperone and contributes to safeguarding Hh by facilitating a soluble multimeric Hh complex [70, 164]	Scube facilitates the release of dually lipid-modified Hh signal in soluble form [70] A heterologous protein complex, i.e., cholesterol anchored Hh-Disp, is transferred to the Scube from Disp in extracellular space for efficient cholesterol-modified Hh secretion [164]
Glioma-associated oncogene (Gli)	Gli is a Krüppel-like transcription factor comprising zinc-finger DNA-binding domains with dual activity [100, 101] Gli family comprises three genes Gli1, Gli2, and Gli3 appear to have similar DNA binding specificities [102, 103] The post-translational proteolytic processing of Gli proteins generates the activator and repressor form of Gli, such as GliA and GliR [105, 107] In response to the Hh signal, Gli-family protein directly regulates Gli1 activation and stimulates numerous Hh target gene [109]
Protein Kinases, such as PKA, DYRK, ULK3, S6K1, AMPK, MEKK1, and Hck	Regulates phosphorylation of Gli-family members and encourages activation of the Hh pathway [116, 146–150] PKA is also involved in Smo phosphorylation [98]
CK1 and Gsk3β	Represented dual role thus it can promote or activate Hh pathway in a context-dependent manner [145] CK1 induces the Smo phosphorylation that is the critical step of Hh signal transduction [98] Gli phosphorylation by CK1 positively regulates Hh pathway activity [145] Gsk3β induces the phosphorylation of Sufu and Gli [17, 19, 125]
E3 ligase complexes, such as Cul1/β-TrCP and E3 ubiquitin-protein ligase Itchy homolog (Itch)	Catalyzes ubiquitination to Gli protein and influences Gli protein activity [150, 151]
Hedgehog-interacting protein (Hip)	Hip is a membrane glycoprotein that interacts with Hh ligands and modulates the Hh downstream signaling [77] Hip sequestrates the Hh ligand, hence acts as a negative regulator of the Hh signaling pathway [48, 77]