The N-Degron Pathway Mediates ER-phagy.

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.

Sphingosylphosphorylcholine blocks ovariectomy-induced bone loss by suppressing Ca2+ /calmodulin-mediated osteoclast differentiation.

Osteoporosis is a disease in which bone mineral density decreases due to abnormal activity of osteoclasts, and is commonly found in post-menopausal women who have decreased levels of female hormones. Sphingosylphosphorylcholine (SPC) is an important biological lipid that can be converted to sphingosine-1-phosphate (S1P) by autotaxin. S1P is known to be involved in osteoclast activation by stimulating osteoblasts, but bone regulation by SPC is not well understood. In this study, we found that SPC strongly inhibits RANKL-induced osteoclast differentiation. SPC-induced inhibitory effects on osteoclast differentiation were not affected by several antagonists of S1P receptors or pertussis toxin, suggesting cell surface receptor independency. However, SPC inhibited RANKL-induced calcineurin activation and subsequent NFATc1 activity, leading to decrease of the expression of Trap and Ctsk. Moreover, we found that bone loss in an experimental osteoporosis mouse model was recovered by SPC injection. SPC also blocked ovariectomy-induced body weight increase and Nfatc1 gene expression in mice. We also found that SPC inhibits RANKL-induced osteoclast differentiation in human macrophages. Since currently available treatments for osteoporosis, such as administration of female hormones or hormone receptor modulators, show serious side effects, SPC has potential as a new agent for osteoporosis treatment.

WIP1, a homeostatic regulator of the DNA damage response, is targeted by HIPK2 for phosphorylation and degradation.

WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR.

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation.

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin-specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation-induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.

The Beneficial Effects of Morusin, an Isoprene Flavonoid Isolated from the Root Bark of Morus.

The root bark of Morus has long been appreciated as an antiphlogistic, diuretic and expectorant drug in Chinese herbal medicine, albeit with barely known targets and mechanisms of action. In the 1970s, the development of analytic chemistry allowed for the discovery of morusin as one of 7 different isoprene flavonoid derivatives in the root bark of Morus. However, the remarkable antioxidant capacity of morusin with the unexpected potential for health benefits over the other flavonoid derivatives has recently sparked scientific interest in the biochemical identification of target proteins and signaling pathways and further clinical relevance. In this review, we discuss recent advances in the understanding of the functional roles of morusin in multiple biological processes such as inflammation, apoptosis, metabolism and autophagy. We also highlight recent in vivo and in vitro evidence on the clinical potential of morusin treatment for multiple human pathologies including inflammatory diseases, neurological disorders, diabetes, cancer and the underlying mechanisms.

Blocking of the IL-33/ST2 Signaling Axis by a Single-Chain Antibody Variable Fragment (scFv) Specific to IL-33 with a Defined Epitope.

Interleukin 33 (IL-33) is an IL-1 family cytokine that plays a central role in immune system by regulating and initiating inflammatory responses. The binding of IL-33 to the suppressor of tumorigenicity 2 (ST2) receptor induces mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) pathways, thereby leading to inflammatory cytokines production in type 2 helper T cells and type 2 innate lymphoid cells. To develop an antibody specific to IL-33 with a defined epitope, we characterized a single-chain antibody variable fragments (scFvs) clone specific to IL-33, C2_2E12, which was selected from a human synthetic library of scFvs using phage display. Affinity (Kd) of C2_2E12 was determined to be 38 nM using enzyme-linked immunosorbent assay. C2_2E12 did not show cross-reactivity toward other interleukin cytokines, including closely related IL-1 family cytokines and unrelated proteins. Mutational scanning analysis revealed that the epitope of IL-33 consisted of residues 149-158 with key residues being L150 and K151 of IL-33. Structural modeling suggested that L150 and K151 residues are important for the interaction of IL-33 with C2_2E12, implicating that C2_2E12 could block the binding of ST2 to IL-33. Pull-down and in-cell assays supported that C2_2E12 can inhibit the IL-33/ST2 signaling axis. These results suggest that the scFv clone characterized here can function as a neutralizing antibody.

Stress Granule Formation Attenuates RACK1-Mediated Apoptotic Cell Death Induced by Morusin.

Stress granules are membraneless organelles composed of numerous components including ribonucleoproteins. The stress granules are characterized by a dynamic complex assembly in response to various environmental stressors, which has been implicated in the coordinated regulation of diverse biological pathways, to exert a protective role against stress-induced cell death. Here, we show that stress granule formation is induced by morusin, a novel phytochemical displaying antitumor capacity through barely known mechanisms. Morusin-mediated induction of stress granules requires activation of protein kinase R (PKR) and subsequent eIF2α phosphorylation. Notably, genetic inactivation of stress granule formation mediated by G3BP1 knockout sensitized cancer cells to morusin treatment. This protective function against morusin-mediated cell death can be attributed at least in part to the sequestration of receptors for activated C kinase-1 (RACK1) within the stress granules, which reduces caspase-3 activation. Collectively, our study provides biochemical evidence for the role of stress granules in suppressing the antitumor capacity of morusin, proposing that morusin treatment, together with pharmacological inhibition of stress granules, could be an efficient strategy for targeting cancer.

ARABIDOPSIS TRITHORAX 4 Facilitates Shoot Identity Establishment during the Plant Regeneration Process.

Plant cells have a remarkable plasticity that allows cellular reprogramming from differentiated cells and subsequent tissue regeneration. Callus formation occurs from pericycle-like cells through a lateral root developmental pathway, and even aerial parts can also undergo the cell fate transition. Pluripotent calli are then subjected primarily to shoot regeneration in in vitro tissue culture. Successful completion of plant regeneration from aerial explants thus entails a two-step conversion of tissue identity. Here we show that a single chromatin modifier, ARABIDOPSIS TRITHORAX 4 (ATX4)/SET DOMAIN GROUP 16, is dynamically regulated during plant regeneration to address proper callus formation and shoot regeneration. The ATX4 protein massively activates shoot identity genes by conferring H3K4me3 deposition at the loci. ATX4-deficient mutants display strong silencing of shoot identity and thus enhanced callus formation. Subsequently, de novo shoot organogenesis from calli is impaired in atx4 mutants. These results indicate that a series of epigenetic reprogramming of tissue identity underlies plant regeneration, and molecular components defining tissue identity can be used as invaluable genetic sources for improving crop transformation efficiency.

SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.

Differential pre-amplification of STR loci for fragmented forensic DNA profiling.

DNA profiling of short tandem repeats (STR) has been successfully used for the identification of individuals in forensic samples, accidents and natural disasters. However, STR profiling of DNA isolated from old crime scenes and damaged biological samples is difficult due to DNA degradation and fragmentation. Here, we show that pre-amplification of STR loci using biotinylated primers for the STR loci is an efficient strategy to obtain STR profiling results from fragmented forensic samples. Analysis of STR loci with longer amplicon sizes is generally hampered, since these relatively long loci are vulnerable to DNA fragmentation. This problem was overcome by using reduced or increased primer concentrations for loci with shorter or longer amplicon sizes, respectively, in our pre-amplification strategy. In addition, pre-amplification of STR loci into two groups of short or long amplicon size increases the efficiency of STR profiling from highly fragmented forensic DNA samples. Therefore, differential pre-amplification of STR loci is an effective way to obtain DNA profiling results from fragmented forensic samples.

Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA).

Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

Optimization of STR locus enrichment for STR profiling of fragmented DNA.

DNA degradation is a major obstacle in gaining an accurate profile with standard DNA typing technology. Although alternative genotyping strategies such as mini-STRs and SNPs have proven to be more successful in profiling degraded DNA, these approaches also have limitations. Here, we show that locus enrichment by hybridization of degraded genomic DNA with an STR locus-specific biotinylated oligonucleotide is a powerful approach to overcome problems in STR typing of highly degraded DNA. An experimental investigation of factors affecting the efficiency of this method indicates that the choice of primer and molar ratio of primers to genomic DNA are critical factors in improving enrichment of the STR locus before genotyping with multiplex kits. In addition, we find that indirect capture rather than direct capture with magnetic beads yields better enrichment efficiency for STR locus enrichments. Using these strategies, we demonstrate an improvement in STR typing of DNA from cultured cells damaged by exposure to sunlight or UV. We suggest that this approach could be applied to highly degraded forensic samples alone or in combination with mini-STRs.

Mdm2 associates with Ras effector NORE1 to induce the degradation of oncoprotein HIPK1.

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.

Tacrolimus-loaded chitosan-based nanoparticles as an efficient topical therapeutic for the effective treatment of atopic dermatitis symptoms.

Atopic dermatitis (AD) is a chronic cutaneous disease with a complex underlying mechanism, and it cannot be completely cured. Thus, most treatment strategies for AD aim at relieving the symptoms. Although corticosteroids are topically applied to alleviate AD, adverse side effects frequently lead to the withdrawal of AD therapy. Tacrolimus (TAC), a calcineurin inhibitor, has been used to treat AD, but its high molecular weight and insolubility in water hinder its skin permeability. Herein, we developed and optimized TAC-loaded chitosan-based nanoparticles (TAC@CNPs) to improve the skin permeability of TAC by breaking the tight junctions in the skin. The prepared nanoparticles were highly loadable and efficient and exhibited appropriate characteristics for percutaneous drug delivery. TAC@CNP was stable for 4 weeks under physiological conditions. CNP released TAC in a controlled manner, with enhanced skin penetration observed. In vitro experiments showed that CNP was non-toxic to keratinocyte (HaCaT) cells, and TAC@CNP dispersed in an aqueous solution was as anti-proliferative as TAC solubilized in a good organic solvent. Importantly, an in vivo AD mouse model revealed that topical TAC@CNP containing ~1/10 of the dose of TAC found in commercially used Protopic® Ointment exhibited similar anti-inflammatory activity to that of the commercial product. TAC@CNP represents a potential therapeutic strategy for the management of AD.

SIRT1 ISGylation accelerates tumor progression by unleashing SIRT1 from the inactive state to promote its deacetylase activity.

ISG15 is an interferon-stimulated ubiquitin-like protein (UBL) with multifaceted roles as a posttranslational modifier in ISG15 conjugation (ISGylation). However, the mechanistic consequences of ISGylation in cancer have not been fully elucidated, largely due to a lack of knowledge on the ISG15 target repertoire. Here, we identified SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, as a new target for ISGylation. SIRT1 ISGylation impairs the association of SIRT1 with its negative regulator, deleted in breast cancer 1 (DBC1), which unleashes SIRT1 from its inactive state and leads to an increase in its deacetylase activity. Importantly, SIRT1 ISGylation promoted lung cancer progression and limited lung cancer cell sensitivity to DNA damage-based therapeutics in vivo and in vitro models. The levels of ISG15 mRNA and protein were significantly higher in lung cancer tissues than in adjacent normal tissues. Accordingly, elevated expression of SIRT1 and ISG15 was associated with poor prognosis in lung cancer patients, a finding that could be translated for lung cancer patient stratification and disease outcome evaluation. Taken together, our findings provide a mechanistic understanding of the regulatory effect of SIRT1 ISGylation on tumor progression and therapeutic efficacy in lung cancer.

WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

SIRT1 negatively regulates the protein stability of HIPK2.

In the present study, we investigated whether a histone deacetylase sirtuin 1 (SIRT1) can regulate the protein stability of homeodomain-interacting protein kinase 2 (HIPK2). We observed the evidence of molecular interaction between SIRT1 and HIPK2. Interestingly, overexpression or pharmacological activation of SIRT1 promoted ubiquitination and the proteasomal degradation of HIPK2 whereas inhibition of SIRT1 activity increased the protein level of HIPK2. Furthermore, a SIRT1 activator decreased the level of HIPK2 acetylation whereas an inhibitor increased the acetylation level. These results suggest that SIRT1 may deacetylate and promote the ubiquitination and subsequent proteasomal degradation of HIPK2.

Two ß-strands of RAGE participate in the recognition and transport of amyloid-ß peptide across the blood brain barrier.

Amyloid-ß (Aß) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aß peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two ß-strands of RAGE participate in the interaction with Aß peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth ß-strands are required for interaction with Aß peptide. Site-directed mutagenesis of amino acids located in the third and eighth ß-strands abolish the interaction of RAGE with Aß peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aß peptide treatment, while a RAGE mutant defective in Aß binding does not. Furthermore, use of peptide for the third ß-strand or a RAGE monoclonal antibody that targets the RAGE-Aß interaction interface inhibited transport of the Aß peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.

L-Type Amino Acid Transporter 1 (LAT1) Promotes PMA-Induced Cell Migration through mTORC2 Activation at the Lysosome.

The mTOR signaling pathway integrates signaling inputs from nutrients, including glucose and amino acids, which are precisely regulated by transporters depending on nutrient levels. The L-type amino acid transporter 1 (LAT1) affects the activity of mTORC1 through upstream regulators that sense intracellular amino acid levels. While mTORC1 activation by LAT1 has been thoroughly investigated in cultured cells, the effects of LAT1 expression on the activity of mTORC2 has scarcely been studied. Here, we provide evidence that LAT1 recruits and activates mTORC2 on the lysosome for PMA-induced cell migration. LAT1 is translocated to the lysosomes in cells treated with PMA in a dose- and time-dependent manner. Lysosomal LAT1 interacted with mTORC2 through a direct interaction with Rictor, leading to the lysosomal localization of mTORC2. Furthermore, the depletion of LAT1 reduced PMA-induced cell migration in a wound-healing assay. Consistent with these results, the LAT1 N3KR mutant, which is defective in PMA-induced endocytosis and lysosomal localization, did not induce mTORC2 recruitment to the lysosome, with the activation of mTORC2 determined via Akt phosphorylation or the LAT1-mediated promotion of cell migration. Taken together, lysosomal LAT1 recruits and activates the mTORC2 complex and downstream Akt for PMA-mediated cell migration. These results provide insights into the development of therapeutic drugs targeting the LAT1 amino acid transporter to block metastasis, as well as disease progression in various types of cancer.

Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53.

Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis.