The N-recognin UBR4 of the N-end rule pathway is targeted to and required for the biogenesis of the early endosome

Kim, Sung Tae,Lee, Yoon Jee,Tasaki, Takafumi,Mun, Su Ran,Hwang, Joonsung,Kang, Min Jueng,Ganipisetti, Srinivasrao,Yi, Eugene C.,Kim, Bo Yeon,Kwon, Yong Tae The Company of Biologists Ltd. 2018 Journal of cell science Vol.131 No.17

<P>The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570 kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca2+-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.</P>

Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule pathway

Cha-Molstad, Hyunjoo,Lee, Su Hyun,Kim, Jung Gi,Sung, Ki Woon,Hwang, Joonsung,Shim, Sang Mi,Ganipisetti, Srinivasrao,McGuire, Terry,Mook-Jung, Inhee,Ciechanover, Aaron,Xie, Xiang-Qun,Kim, Bo Yeon,Kwon, Informa UK (TaylorFrancis) 2018 AUTOPHAGY Vol.14 No.2

<P>In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62 (sequestosome 1), and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons (Nt-Lys, Nt-His, Nt-Trp, Nt-Phe, and Nt-Tyr) of the N-end rule pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone HSPA5/GRP78/BiP. Upon N-end rule interaction with the Nt-Arg of arginylated HSPA5 (R-HSPA5), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatory mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.</P>

Mechanism of the natural product moracin-O derived MO-460 and its targeting protein hnRNPA2B1 on HIF-1α inhibition

성낙균,김혜민,Yukihiro Asami,김동현,조양래,Ravi Naik,장예린,장구식,한호진,Srinivas Rao Ganipisetti,차현주,황준성,이경호,고성균,장재혁,류인자,권용태,이경상,Hiroyuki Osada,이경,김보연,안종석 생화학분자생물학회 2019 Experimental and molecular medicine Vol.51 No.-

Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycinerich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3’-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxiainduced tumor survival and thus offer an avenue for the development of novel anticancer therapies.

The N-Degron Pathway Mediates ER-phagy

Ji, Chang Hoon,Kim, Hee Yeon,Heo, Ah Jung,Lee, Su Hyun,Lee, Min Ju,Kim, Su Bin,Srinivasrao, Ganipisetti,Mun, Su Ran,Cha-Molstad, Hyunjoo,Ciechanover, Aaron,Choi, Cheol Yong,Lee, Hee Gu,Kim, Bo Yeon,Kw Elsevier 2019 Molecular Cell Vol.75 No.5

<P><B>Summary</B></P> <P>The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Here, we show that the N-degron pathway mediates ER-phagy. This autophagic degradation initiates when the transmembrane E3 ligase TRIM13 (also known as RFP2) is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62 (also known as sequestosome-1), whose complex undergoes oligomerization. The oligomerization is induced when the ZZ domain of p62 is bound by the N-terminal arginine (Nt-Arg) of arginylated substrates. Upon activation by the Nt-Arg, oligomerized TRIM13-p62 complexes are separated along with the ER compartments and targeted to autophagosomes, leading to lysosomal degradation. When protein aggregates accumulate within the ER lumen, degradation-resistant autophagic cargoes are co-segregated by ER membranes for lysosomal degradation. We developed synthetic ligands to the p62 ZZ domain that enhance ER-phagy for ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The autophagic adaptor p62 mediates autophagic degradation of the ER (ER-phagy) </LI> <LI> The ER membrane E3 ligase TRIM13 is a ubiquitin-dependent ER-phagy receptor to p62 </LI> <LI> N-terminal arginylation is an ER-phagy degron via binding to the ZZ domain of p62 </LI> <LI> p62-TRIM13-Nt-Arg circuit mediates ER protein quality control and homeostasis </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Mechanism of the natural product moracin-O derived MO-460 and its targeting protein hnRNPA2B1 on HIF-1α inhibition

Soung, Nak-Kyun,Kim, Hye-Min,Asami, Yukihiro,Kim, Dong Hyun,Cho, Yangrae,Naik, Ravi,Jang, Yerin,Jang, Kusic,Han, Ho Jin,Ganipisetti, Srinivas Rao,Cha-Molstad, Hyunjoo,Hwang, Joonsung,Lee, Kyung Ho,Ko, Nature Publishing Group UK 2019 Experimental and molecular medicine Vol.51 No.2

<▼1><P>Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3’-untranslated region of <I>HIF-1α</I> mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.</P></▼1><▼2><P><B>Cancer: How a plant metabolite analog suppresses tumor growth</B></P><P>A synthetic analog of a chemical found in fruit suppresses tumor growth by targeting an RNA-binding protein (hnRNPA2B1) and preventing the production of a pro-cancer regulatory factor. Nak-Kyun Soung from the Korea Research Institute of Bioscience and Biotechnology, Cheongju, South Korea, and coworkers built on their previous discovery that a compound derived from a medicinal plant metabolite can suppress the activity of hypoxia-inducible factor-1α (HIF-1α). This protein, which is involved in many aspects of cancer biology, is activated in the low-oxygen microenvironments found inside tumors. The researchers show that the compound binds to a protein that helps with the conversion of HIF-1α–encoding RNA transcripts into HIF-1α proteins. Liver cancer cells treated with the compound grew slowly and produced less HIF-1α under both normal and low-oxygen culture conditions, highlighting the potential of this anti-cancer strategy.</P></▼2>