Chemical Protein Degradation Targeting Non-Functional Binding Sites

Synfacts ◽  
2019 ◽  
Vol 15 (09) ◽  
pp. 1070
2020 ◽  
Vol 295 (50) ◽  
pp. 17227-17240 ◽  
Author(s):  
Liu Liu ◽  
Arti B. Dumbrepatil ◽  
Angela S. Fleischhacker ◽  
E. Neil G. Marsh ◽  
Stephen W. Ragsdale

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.


2021 ◽  
Vol 71 (3) ◽  
pp. 161-176
Author(s):  
Mladen Koravović ◽  
Gordana Tasić ◽  
Milena Rmandić ◽  
Bojan Marković

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.


2020 ◽  
Author(s):  
Liu Liu ◽  
Arti B. Dumbrepatil ◽  
Angela S. Fleischhacker ◽  
E. Neil G. Marsh ◽  
Stephen W. Ragsdale

ABSTRACTHeme oxygenase-2 (HO2) and −1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2’s role in maintaining heme homeostasis, paving the way for future investigation into HO2’s pathophysiological role in heme deficiency response.


1999 ◽  
Vol 274 (40) ◽  
pp. 28690-28696 ◽  
Author(s):  
Gordon S. Huggins ◽  
Michael T. Chin ◽  
Nicholas E. S. Sibinga ◽  
Shwu-Luan Lee ◽  
Edgar Haber ◽  
...  

2014 ◽  
Vol 112 (1) ◽  
pp. 106-111 ◽  
Author(s):  
Jee-Young Mock ◽  
Justin William Chartron ◽  
Ma’ayan Zaslaver ◽  
Yue Xu ◽  
Yihong Ye ◽  
...  

BCL2-associated athanogene cochaperone 6 (Bag6) plays a central role in cellular homeostasis in a diverse array of processes and is part of the heterotrimeric Bag6 complex, which also includes ubiquitin-like 4A (Ubl4A) and transmembrane domain recognition complex 35 (TRC35). This complex recently has been shown to be important in the TRC pathway, the mislocalized protein degradation pathway, and the endoplasmic reticulum-associated degradation pathway. Here we define the architecture of the Bag6 complex, demonstrating that both TRC35 and Ubl4A have distinct C-terminal binding sites on Bag6 defining a minimal Bag6 complex. A crystal structure of the Bag6–Ubl4A dimer demonstrates that Bag6–BAG is not a canonical BAG domain, and this finding is substantiated biochemically. Remarkably, the minimal Bag6 complex defined here facilitates tail-anchored substrate transfer from small glutamine-rich tetratricopeptide repeat-containing protein α to TRC40. These findings provide structural insight into the complex network of proteins coordinated by Bag6.


2019 ◽  
Author(s):  
Wen Xiong ◽  
Tuo-Xian Tang ◽  
Evan Littleton ◽  
Arba Karcini ◽  
Iulia M. Lazar ◽  
...  

AbstractTom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate [PtdIns5P], which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.


2021 ◽  
Author(s):  
Colten K Lankford ◽  
Jon C Houtman ◽  
Sheila A Baker

Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. Isothermal titration calorimetry demonstrated that recombinant 14-3-3 binds all three phospho-peptides with low micromolar affinity. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two binding sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding sites is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.


1998 ◽  
Vol 4 (S2) ◽  
pp. 978-979
Author(s):  
Martin Kessel ◽  
Fabienne Beuron ◽  
Frank Booy ◽  
Eva Kocsis ◽  
Michael Maurizi ◽  
...  

ATP-dependent proteases play a major role in regulatory protein degradation in both prokaryotic and eukaryotic cells. ATP-dependent proteases in E. coli fall into two classes. The first class requires the interaction of structurally separate proteases with an ATPase, whereas in the second class both the protease and ATPase are formed from regions of the same polypeptide chain. We have studied the structure of several of these protein degrading complexes in E. coli and have found a remarkable similarity in the architecture of these macromolecular assemblies.The prototypical protease of the first class has as its proteolytic component ClpP, a 14 subunit (MW 21,500) complex arranged as two lOnm-diameter stacked rings of seven subunits each. ClpP can interact with either one of two ATPases, ClpA or ClpX, each with unique substrate specificity. ClpA has two ATP-binding sites per subunit (MW 84,000), and its subunits are arranged as a 13nm (diameter) hexameric ring (MW -500,000).


1982 ◽  
Vol 101 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Geneviève Grizard ◽  
Daniel Boucher ◽  
Jean Hermabessière ◽  
Jean Grizard

Abstract. Binding and degradation of human chorionic gonadotrophin (hCG) to testicular tissue obtained by biopsy from 9 men with gonadal disorders were investigated. Vacant hCG receptors were assayed in partially purified testicular homogenates using [125I]hCG (radio-iodinated with chloramine T). Degradation of [125I]hCG during exposure to human testicular preparations was measured in terms of the ability of supernatants to specifically bind to rat testicular receptors. Binding of [125I]hCG was time and temperature dependent. At 37°C, a maximum was reached at 8 h. It was also found to be a saturable process with respect to homogenate and hormone concentrations. Association constants and number of binding sites determined in 9 men, using Scatchard plot and saturation curve analysis ranged, respectively, from 0.2 to 1.8 × 1010m−1 and from 92 to 3427 fmol/g testis or 7 to 380 fmol/mg protein. Degradation of [125I]hCG increased with temperature and time of exposure to human testicular homogenate. It increased also with increasing human testicular homogenate concentration and substrate concentrations. For a similar concentration of [125I]hCG, per cent of degraded hormone ranged from 32 to 57, according to the subjects. These results show that human testicular homogenates are capable of binding and degrading hCG in vitro. Biological and physiological implications of degradation for hormone binding are discussed.


1999 ◽  
Vol 81 (03) ◽  
pp. 428-435 ◽  
Author(s):  
Michael Green ◽  
Philip LoGrasso ◽  
Brian Boettcher ◽  
Edwin Madison ◽  
Linda Curtiss ◽  
...  

SummaryLipoprotein(a) [Lp(a)] is associated with atherosclerosis and with disease processes involving thrombosis. Lp(a) contains apoprotein (a) [apo(a)], which has a sequence highly homologous to plasminogen. Hence, Lp(a) binds directly to extracellular matrix, cellular plasminogen receptors and fibrin(ogen) and competes for the binding of plasminogen to these regulatory surfaces. These interactions may contribute to the proatherothrombogenic consequences of high Lp(a) levels. These interactions are mediated by lysine binding sites (LBS). Therefore, we examined the role of apo(a) kringle IV-10 [the only apo(a) kringle demonstrated to exhibit lysine binding activity in the intact lipoprotein] in the interaction of Lp(a) with these regulatory molecules. We have compared directly apo(a) KIV-10 with plasminogen K4 to examine whether these highly structurally homologous kringle modules are also functionally homologous. Futhermore, because the plasminogen K5-protease domain (K5-PD) binds directly to fibrin, we have also examined the ability of this plasminogen fragment to inhibit the interaction of Lp(a) with these regulatory molecules and with extracellular matrix. Apo(a) KIV-10 competed effectively for the binding of 125I-Lp(a) to these surfaces but was less effective than either intact Lp(a), plasminogen K4 or plasminogen. Plasminogen K5-PD was a better competitor than apo(a) KIV-10 for 125I-Lp(a) binding to the representative extra-cellular matrix, Matrigel, and to plasmin-treated fibrinogen. In contrast, plasminogen K5-PD did not compete for the interaction of Lp(a) with cells, although it effectively competed for plasminogen binding. These results suggest that Lp(a) recognizes sites in all of the regulatory molecules that are also recognized by apo(a) KIV-10 and that Lp(a) recognizes sites in extracellular matrix and in plasmin-modified fibrinogen that also are recognized by plasminogen K5-PD. Thus, the interaction of Lp(a) with cells is clearly distinct from that with extracellular matrix and with plasmin-treated fibrinogen and the recognition sites within Lp(a) and plasminogen for these regulatory molecules are not identical.Portions of this manuscript were presented at the 69th Meeting of the American Heart Association, New Orleans, LA 1996.


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