RCHY1 and OPTN are required for melanophagy, selective autophagy of melanosomes.

Melanosomes are specific organelles dedicated to melanin synthesis and accumulation in melanocytes. Autophagy is suggestively involved in melanosome degradation, although the potential underlying molecular mechanisms remain elusive. In selective autophagy, autophagy receptors and E3-ligases are the key factors conferring cargo selectivity. In B16F10 cells, ß-mangostin efficiently induced melanosome degradation without affecting other organelles such as mitochondria, peroxisomes, and the endoplasmic reticulum. Among various autophagy receptors, optineurin (OPTN) contributes TANK-binding kinase 1 (TBK1)-dependently to melanosome degradation and its knockdown inhibited ß-mangostin-mediated melanosome degradation. OPTN translocation to melanosomes was dependent on its ubiquitin-binding domain. Moreover, OPTN-mediated TBK1 activation and subsequent TBK1-mediated S187 OPTN phosphorylation were essential for melanosome degradation. ß-mangostin increased K63-linked melanosome ubiquitination. Finally, the E3-ligase RCHY1 knockdown inhibited the melanosome ubiquitination required for OPTN- and TBK1-phosphorylation as well as melanosome degradation. This study suggests that melanophagy, melanosome-selective autophagy, contributes to melanosome degradation, and OPTN and RCHY1 are an essential autophagy receptor and a E3-ligase, respectively, conferring cargo selectivity in melanophagy.

Consideration of SHP-1 as a Molecular Target for Tumor Therapy.

Abnormal activation of receptor tyrosine kinases (RTKs) contributes to tumorigenesis, while protein tyrosine phosphatases (PTPs) contribute to tumor control. One of the most representative PTPs is Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1), which is associated with either an increased or decreased survival rate depending on the cancer type. Hypermethylation in the promoter region of PTPN6, the gene for the SHP-1 protein, is a representative epigenetic regulation mechanism that suppresses the expression of SHP-1 in tumor cells. SHP-1 comprises two SH2 domains (N-SH2 and C-SH2) and a catalytic PTP domain. Intramolecular interactions between the N-SH2 and PTP domains inhibit SHP-1 activity. Opening of the PTP domain by a conformational change in SHP-1 increases enzymatic activity and contributes to a tumor control phenotype by inhibiting the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway. Although various compounds that increase SHP-1 activation or expression have been proposed as tumor therapeutics, except sorafenib and its derivatives, few candidates have demonstrated clinical significance. In some cancers, SHP-1 expression and activation contribute to a tumorigenic phenotype by inducing a tumor-friendly microenvironment. Therefore, developing anticancer drugs targeting SHP-1 must consider the effect of SHP-1 on both cell biological mechanisms of SHP-1 in tumor cells and the tumor microenvironment according to the target cancer type. Furthermore, the use of combination therapies should be considered.

Amorphigenin from Amorpha fruticosa L. Root Extract Induces Autophagy-Mediated Melanosome Degradation in mTOR-Independent- and AMPK-Dependent Manner.

In this study, we investigated the depigmentation effect of Amorpha fruticosa L. root extract (RE), an herbal medicine. A. fruticosa RE significantly induced depigmentation in α-MSH-treated B16F10 cells at noncytotoxic concentrations. Further, the RE decreased the protein levels of the melanosomal proteins Tyr and Pmel without decreasing their transcript levels. We found that MG132, a proteasome complex inhibitor, was unable to rescue the protein levels, but PepA/E-64D (a lysosomal enzyme inhibitor), 3-MA (a representative autophagy inhibitor), and ATG5 knockdown effectively rescued the protein levels and inhibited the depigmentation effect following RE treatment. Among rotenoids, amorphigenin composed in the RE was identified as a functional chemical that could induce depigmentation; whereas rapamycin, an mTOR inhibitor and a nonselective autophagy inducer, could not induce depigmentation, and amorphigenin effectively induced depigmentation through the degradation of melanosomal proteins. Amorphigenin activated AMPK without affecting mTOR, and knockdown of AMPK offset the whitening effect through degradation of melanosome proteins by amorphigenin. Results from this study suggested that amorphigenin can induce degradation of the melanosome through an AMPK-dependent autophagy process, and has the potential to be used as a depigmentation agent for the treatment of hyperpigmentation.

The Function of N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Negative Regulator in Tumor Cell Metastasis.

N-myc downstream-regulated gene 2 (NDRG2) is a tumor-suppressor gene that suppresses tumorigenesis and metastasis of tumors and increases sensitivity to anti-cancer drugs. In this review, we summarize information on the clinicopathological characteristics of tumor patients according to NDRG2 expression in various tumor tissues and provide information on the metastasis inhibition-related cell signaling modulation by NDRG2. Loss of NDRG2 expression is a prognostic factor that correlates with TNM grade and tumor metastasis and has an inverse relationship with patient survival in various tumor patients. NDRG2 inhibits cell signaling, such as AKT-, NF-κB-, STAT3-, and TGF-ß-mediated signaling, to induce tumor metastasis, and induces activation of GSK-3ß which has anti-tumor effects. Although NDRG2 operates as an adaptor protein to mediate the interaction between kinases and phosphatases, which is essential in regulating cell signaling related to tumor metastasis, the molecular mechanism of NDRG2 as an adapter protein does not seem to be fully elucidated. This review aims to assist the research design regarding NDRG2 function as an adaptor protein and suggests NDRG2 as a molecular target to inhibit tumor metastasis and improve the prognosis in tumor patients.

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chamber's equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

Cryo-EM structures of GroEL:ES2 with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

The GroEL/GroES chaperonin system assists the folding of many proteins, through conformational transitions driven by ATP hydrolysis. Although structural information about bullet-shaped GroEL:ES1 complexes has been extensively reported, the substrate interactions of another functional complex, the football-shaped GroEL:ES2, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES2 complexes with a chemically denatured substrate, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Our structures demonstrate that native-like folded RuBisCO density is captured at the lower part of the GroEL chamber and that GroEL's bulky hydrophobic residues Phe281, Tyr360, and Phe44 contribute to direct contact with RuBisCO density. In addition, our analysis found that GroEL:ES2 can be occupied by two substrates simultaneously, one in each chamber. Together, these observations provide insights to the football-shaped GroEL:ES2 complex as a functional state to assist the substrate folding with visualization.

N-myc Downstream-Regulated Gene 2 (NDRG2) Function as a Positive Regulator of Apoptosis: A New Insight into NDRG2 as a Tumor Suppressor.

N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene that increases tumor sensitivity to anticancer drugs, slows tumor progression, and inhibits metastasis. NDRG2 is suppressed in various aggressive tumor positions, whereas NDRG2 expression is associated with patient prognosis, such as an improved survival rate. In this review, we summarize the tumor suppressor mechanism of NDRG2 and provide information on the function of NDRG2 concerning the susceptibility of cells to apoptosis. NDRG2 increases the susceptibility to apoptosis in various physiological environments of cells, such as development, hypoxia, nutrient deprivation, and cancer drug treatment. Although the molecular and cell biological mechanisms of NDRG2 have not been fully elucidated, we provide information on the mechanisms of NDRG2 in relation to apoptosis in various environments. This review can assist the design of research regarding NDRG2 function and suggests the potential of NDRG2 as a molecular target for cancer patients.