Protein–Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing

Zhang, Xiaoyu; Xu, Zihan; Moumin, Dina S; Ciulla, Daniel A.; Owen, Timothy S.; Mancusi, Rebecca; Giner, José‐Luis; Wang, Chunyu; Callahan, Brian P.

doi:10.1021/acs.bioconjchem.9b00550

Cited by 13 publications

(29 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Accordingly, cholesterol ligation can occur in vitro and in cells when the Hh N-terminus is replaced by fluorescent proteins or simply by a His-tag. [18][19][20][21][22] However, the molecular details of the SRR that facilitate cholesterol transfer remain unknown. The SRR, which spans 50-100 residues in different species, bears no clear homology to known proteins in any domain of life.…”

mentioning

confidence: 99%

Dual roles of the sterol recognition region in Hedgehog protein modification

et al. 2020

View full text Add to dashboard Cite

Nature provides a number of mechanisms to encode dynamic information in biomolecules. In metazoans, there exist rare chemical modifications that occur in entirely unique regimes. One such example occurs in the Hedgehog (Hh) morphogens, proteins singular across all domains of life for the nature of their covalent ligation to cholesterol. The isoform-and context-specific efficiency of this ligation profoundly impacts the activity of Hh morphogens and represents an unexplored facet of Hh ligand-dependent cancers. To elucidate the chemical mechanism of this modification, we have defined roles of the uncharacterized sterol recognition region (SRR) in Hh proteins. We use a combination of sequence conservation, directed mutagenesis, and biochemical assays to specify residues of the SRR participate in cellular and biochemical aspects of Hh cholesterolysis. Our investigations offer a functional portrait of this region, providing opportunities to identify parallel reactivity in nature and a template to design tools in chemical biology.

show abstract

mentioning

confidence: 99%

Dual roles of the sterol recognition region in Hedgehog protein modification

et al. 2020

View full text Add to dashboard Cite

show abstract

“…With the resulting approximation, we created a full lipid bilayer containing 100 POPC molecules in each leaflet with~20% cholesterol content (24 cholesterol molecules in each leaflet), approximating the composition of cholesterol-rich microdomains in the Golgi and endoplasmic reticulum [41][42][43][44]. Several previous studies have demonstrated that Hedge domain residues N-terminal to C198 are not required for cholesterolysis [45][46][47][48]; therefore, we modeled the N-terminal portion of hSHH (C24-G197) as a G197-C198 thioester preceded by a S195-G196 dipeptide for computational simplicity. To prepare the system for MD simulations, we solvated the protein, neutralized charges with NaCl (0.15 M), and adjusted the system to physiological pH (7.4).…”

Section: Simulations Place a Hshh Hog Protein On The Membranementioning

confidence: 99%

Hedgehog proteins create a dynamic cholesterol interface

et al. 2021

View full text Add to dashboard Cite

During formation of the Hedgehog (Hh) signaling proteins, cooperative activities of the Hedgehog INTein (Hint) fold and Sterol Recognition Region (SRR) couple autoproteolysis to cholesterol ligation. The cholesteroylated Hh morphogens play essential roles in embryogenesis, tissue regeneration, and tumorigenesis. Despite the centrality of cholesterol in Hh function, the full structure of the Hint-SRR (“Hog”) domain that attaches cholesterol to the last residue of the active Hh morphogen remains enigmatic. In this work, we combine molecular dynamics simulations, photoaffinity crosslinking, and mutagenesis assays to model cholesterolysis intermediates in the human Sonic Hedgehog (hSHH) protein. Our results provide evidence for a hydrophobic Hint-SRR interface that forms a dynamic, non-covalent cholesterol-Hog complex. Using these models, we suggest a unified mechanism by which Hh proteins can recruit, sequester, and orient cholesterol, and offer a molecular basis for the effects of disease-causing hSHH mutations.

show abstract

“…Various studies reported that certain OSBPs can be involved in different types of human tumors [ 26 ]. Moreover, Zhang et al [ 27 ] described the covalent conjugation of protein and DNA with sterols serving as molecular linkers using Hedgehog autoprocessing. More recently, the non-covalent conjugations of bovine serum albumin with β-sitosterol and stigmasterol were reported to compete against glycation [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

Food Protein Sterylation: Chemical Reactions between Reactive Amino Acids and Sterol Oxidation Products under Food Processing Conditions

Nzekoue

Henle

Caprioli

et al. 2020

Foods

View full text Add to dashboard Cite

Sterols, especially cholesterol and phytosterols, are important components of food lipids. During food processing, such as heating, sterols, like unsaturated fatty acids, can be oxidized. Protein modification by secondary products of lipid peroxidation has recently been demonstrated in food through a process called lipation. Similarly, this study was performed to assess, for the first time, the possibility of reactions between food proteins and sterol oxidation products in conditions relevant for food processing. Therefore, reaction models consisting of oxysterol (cholesterol 5α,6α-epoxide) and reactive amino acids (arginine, lysine, and methionine) were incubated in various conditions of concentration (0–8 mM), time (0–120 min), and temperature (30–180 °C). The identification of lysine adducts through thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) with a diode array detector (DAD), and electrospray ionization (ESI) mass spectrometry (MS) evidenced a reaction with lysine. Moreover, the HPLC-ESI with tandem mass spectrometry (MS/MS) analyses allowed observation of the compound, whose mass to charge ratio m/z 710.5 and fragmentation patterns corresponded to the reaction product [M + H]+ between cholesterol-5α,6α-epoxide and the ε-amino-group of Nα-benzoylglycyl-l-lysine. Moreover, kinetic studies between Nα-benzoylglycyl-l-lysine as a model for protein-bound lysine and cholesterol 5α,6α-epoxide were performed, showing that the formation of lysine adducts strongly increases with time, temperature, and oxysterol level. This preliminary study suggests that in conditions commonly reached during food processing, sterol oxidation products could react covalently with protein-bound lysine, causing protein modifications.

show abstract

Protein–Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing

Cited by 13 publications

References 39 publications

Dual roles of the sterol recognition region in Hedgehog protein modification

Dual roles of the sterol recognition region in Hedgehog protein modification

Hedgehog proteins create a dynamic cholesterol interface

Food Protein Sterylation: Chemical Reactions between Reactive Amino Acids and Sterol Oxidation Products under Food Processing Conditions

Contact Info

Product

Resources

About