The Testis Is a Primary Factor That Contributes to Epigenetic Modifications in the Ovaries of the Protandrous Black Porgy, Acanthopagrus schlegelii.

In most hermaphroditic fish, the sexual phase of the gonad responds to external stimuli so that only one sex remains functional while the other sex becomes dormant. However, protandrous black porgy are male during their first two reproductive cycles. Estradiol (E2)-induced female growth results in a transient and immature female, and the sexual phase reverts from female to male after E2 is withdrawn. Conversely, excising the testis results in a precocious female when performed during the second reproductive cycle. We used these characteristics to study epigenetic modifications of cyp19a1a promoter in black porgy. Our results showed that higher levels of gonadotropins receptors were observed in testis than in ovary during the alteration of sexual phase from induced femaleness to maleness, and hCG treatment did not stimulate ovarian gene expression in male (1-yr-old maleness) and female phase (testis excision-induced femaleness) fish. The cyp19a1a promoter exhibited tissue- and lineage-specific methylation patterns. The follicle cells in the ovary had a hypomethylated (0%-20%) cyp19a1a promoter region. In the ovary, the first sign of female phase decision was decreased methylation levels and increased numbers of hypomethylated clones of cyp19a1a promoter during the natural sex change process. Similar methylation patterns were observed in the testis-removed ovary 1 mo after surgery, with no histological difference between the sham and the testis-removed fish. Conversely, there was no increase in methylation levels of cyp19a1a promoter in E2-fed fish. These results suggest that in the digonic gonad of black porgy, the testis is the primary tissue that affects epigenetics of the ovary.

Role of HERP and a HERP-related protein in HRD1-dependent protein degradation at the endoplasmic reticulum.

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of retrotranslocation is unclear. Here, we use several lumenal ERAD substrates targeted for degradation by the ubiquitin ligase HRD1 including SHH (sonic hedgehog) and NHK (null Hong Kong α1-antitrypsin) to study the geometry, organization, and regulation of the HRD1-containing ERAD machinery. We report a new HRD1-associated membrane protein named HERP2, which is homologous to the previously identified HRD1 partner HERP1. Despite sequence homology, HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress. We find that these proteins are required for efficient degradation of both glycosylated and nonglycosylated SHH proteins as well as NHK. In cells depleted of HERPs, SHH proteins are largely trapped inside the ER with a fraction of the stabilized SHH protein bound to the HRD1-SEL1L ligase complex. Ubiquitination of SHH is significantly attenuated in the absence of HERPs, suggesting a defect in retrotranslocation. Both HERP proteins interact with HRD1 through a region located in the cytosol. However, unlike its homolog in Saccharomyces cerevisiae, HERPs do not regulate HRD1 stability or oligomerization status. Instead, they help recruit DERL2 to the HRD1-SEL1L complex. Additionally, the UBL domain of HERP1 also seems to have a function independent of DERL2 recruitment in ERAD. Our studies have revealed a critical scaffolding function for mammalian HERP proteins that is required for forming an active retrotranslocation complex containing HRD1, SEL1L, and DERL2.

Novel Nrf2/ARE activator, trans-Coniferylaldehyde, induces a HO-1-mediated defense mechanism through a dual p38α/MAPKAPK-2 and PK-N3 signaling pathway.

The induction of detoxifying enzymes and antioxidant proteins by chemopreventive agents protects cells from oxidizing substances capable of damaging DNA integrity and initiating carcinogenesis. Coniferyl aldehyde, a naturally occurring substance, has been found in many foods and edible plants. We and others previously demonstrated that trans-coniferylaldehyde (t-CA) has potential antimutagenic and antioxidant properties. However, the mechanism underlying its Nrf2-mediated antioxidant effect remains largely unknown. In the present study, we demonstrated that t-CA significantly stimulated antioxidant-responsive element (ARE)-driven luciferase activity in a cell model and increased the expression of ARE-dependent detoxifying/antioxidant genes and their protein products in vitro and in vivo. The detoxifying/antioxidant genes activated by t-CA, especially heme oxygenase-1 (HO-1), were found to be involved in its cytoprotective effects against oxidative stress and cell injuries elicited by carcinogens tert-butylhydroperoxide and arecoline. Furthermore, the t-CA-induced phosphorylation and nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) played a crucial role in this ARE-mediated cellular defense. Moreover, we found that p38 MAPK and protein kinase C (PKC) signaling pathways participated in the t-CA-induced, Nrf2-mediated cytoprotective effect. Among them, p38α/MAPKAPK-2 and an atypical PKC, PK-N3, were critical for the activation of the Nrf2/HO-1 axis by t-CA. In conclusion, we demonstrated for the first time that t-CA attenuates carcinogen-induced oxidative stress by activating Nrf2 via p38α/MAPKAPK-2- and PK-N3-dependent signaling pathways. In addition, t-CA increased the level of Nrf2-mediated detoxifying/antioxidant proteins in vivo, suggesting that t-CA may have potential for use in the management of carcinogenesis and meriting further investigation.

Derlin2 protein facilitates HRD1-mediated retro-translocation of sonic hedgehog at the endoplasmic reticulum.

Endoplasmic reticulum-associated degradation (ERAD) is an important system that eliminates misfolded proteins from the ER. Three derlins have been implicated in this process, but their precise function remains unknown. In this study, we report that although both derlin1 and derlin2 are capable of binding the ERAD-specific ubiquitin ligase HRD1, they associate with the HRD1-containing complex with different affinities. Accordingly, these derlins have nonredundant functions in ERAD with derlin2 being an essential functional partner for HRD1-mediated ERAD of SHH and NHK. We show that derlin2, but not derlin1 or derlin3, is required for ERAD of both glycosylated and nonglycosylated SHH, as well as NHK. Derlin2 appears to act at a post-targeting step for HRD1-dependent retro-translocation. Without derlin2, the assembly of HRD1 into a functional retro-translocation homo-oligomer proceeds normally, and substrate targeting to the HRD1 complex also occurs. However, the ERAD substrate SHH-C is largely trapped inside the ER lumen. These observations raise the possibility that derlin2 may regulate the movement of substrates through the HRD1-containing retro-translocon. Our study is the first to report that derlin2 functions with HRD1 in ERAD of certain substrates independent of their glycosylation status. The mammalian ERAD system may require multiple derlins that each functions with a distinct E3 partner to eliminate a specific subset of substrates. This is different from the model in Saccharomyces cerevisiae, in which Hrd1p alone is sufficient for retro-translocation.

Lightweight Deep Neural Network for Joint Learning of Underwater Object Detection and Color Conversion.

Underwater image processing has been shown to exhibit significant potential for exploring underwater environments. It has been applied to a wide variety of fields, such as underwater terrain scanning and autonomous underwater vehicles (AUVs)-driven applications, such as image-based underwater object detection. However, underwater images often suffer from degeneration due to attenuation, color distortion, and noise from artificial lighting sources as well as the effects of possibly low-end optical imaging devices. Thus, object detection performance would be degraded accordingly. To tackle this problem, in this article, a lightweight deep underwater object detection network is proposed. The key is to present a deep model for jointly learning color conversion and object detection for underwater images. The image color conversion module aims at transforming color images to the corresponding grayscale images to solve the problem of underwater color absorption to enhance the object detection performance with lower computational complexity. The presented experimental results with our implementation on the Raspberry pi platform have justified the effectiveness of the proposed lightweight jointly learning model for underwater object detection compared with the state-of-the-art approaches.

Discovery and development of a novel N-(3-bromophenyl)-{[(phenylcarbamoyl)amino]methyl}-N-hydroxythiophene-2-carboximidamide indoleamine 2,3-dioxygenase inhibitor using knowledge-based drug design.

Indoleamine 2,3-dioxygenase-1 (IDO1) is a potential target for the next generation of cancer immunotherapies. We describe the development of two series of IDO1 inhibitors incorporating a N-hydroxy-thiophene-carboximidamide core generated by knowledge-based drug design. Structural modifications to improve the cellular activity and pharmacokinetic (PK) properties of the compounds synthesized, including extension of the side chain of the N-hydroxythiophene-2-carboximidamide core, resulted in compound 27a, a potent IDO1 inhibitor which demonstrated significant (51%) in vivo target inhibition on IDO1 in a human SK-OV-3 ovarian xenograft tumor mouse model. This strategy is expected to be applicable to the discovery of additional IDO1 inhibitors for the treatment of other diseases susceptible to modulation of IDO1.

Proline in transmembrane domain of type II protein DPP-IV governs its translocation behavior through endoplasmic reticulum.

A transmembrane domain (TMD) at the N-terminus of a membrane protein is a signal sequence that targets the protein to the endoplasmic reticulum (ER) membrane. Proline is found more frequently in TM helices compared to water-soluble helices. To investigate the effects of proline on protein translocation and integration in mammalian cells, we made proline substitutions throughout the TMD of dipeptidyl peptidase IV, a type II membrane protease with a single TMD at its N-terminus. The proteins were expressed and their capacities for targeting and integrating into the membrane were measured in both mammalian cells and in vitro translation systems. Three proline substitutions in the central region of the TMD resulted in various defects in membrane targeting and/or integration. The replacement of proline with other amino acids of similar hydrophobicity rescued both the translocation and anchoring defects of all three proline mutants, indicating that conformational change caused by proline is a determining factor. Increasing hydrophobicity of the TMD by replacing other residues with more hydrophobic residues also effectively reversed the translocation and integration defects. Intriguingly, increasing hydrophobicity at the C-terminal end of the TMD rescued much more effectively than it did at the N-terminal end. Thus, the effect of proline on translocation and integration of the TMD is not determined solely by its conformation and hydrophobicity, but also by the location of proline in the TMD, the location of highly hydrophobic residues, and the relative position of the proline to other proline residues in the TMD.

Xanthine Derivatives Reveal an Allosteric Binding Site in Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2).

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.

(2S,4S)-1-[2-(1,1-dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino)acetyl]-4-fluoro-pyrrolidine-2-carbonitrile: a potent, selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV.

A series of 2-[3-[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]-based DPP-IV inhibitors with various monocyclic amines were synthesized. The structure-activity relationships (SAR) led to the discovery of potent DPP-IV inhibitors, having IC(50) values of <100 nM with excellent selectivity over the closely related enzymes, DPP-II, DPP8, DPP9 and FAP (IC(50)>20 microM). Of these compounds, the analogues 12a, 12h and 12i exhibited a long-lasting ex vivo DPP-IV inhibition in rats.

Rational design and synthesis of potent and long-lasting glutamic acid-based dipeptidyl peptidase IV inhibitors.

A series of (2S)-cyanopyrrolidines with glutamic acid derivatives at the P2 site have been prepared and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV). The structure-activity relationships (SAR) led to the discovery of potent 3-substituted glutamic acid analogues, providing enhanced chemical stability and excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. Compound 13f exhibited the ability to both significantly decrease the glucose excursion and inhibit plasma DPP-IV activity.

Novel trans-2-aryl-cyclopropylamine analogues as potent and selective dipeptidyl peptidase IV inhibitors.

A series of trans-2-aryl-cyclopropylamine derived compounds were synthesized and evaluated their biological activities against DPP-IV. The structure-activity relationships (SAR) led to the discovery of novel series of DPP-IV inhibitors, having IC(50) values of <100 nM with excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. The studies identified a potent and selective DPP-IV inhibitor 24b, which exhibited the ability to both significantly inhibit plasma DPP-IV activity in rats and improve glucose tolerance in lean mice and diet induced obese mice.

Multi-Scale Deep Residual Learning-Based Single Image Haze Removal via Image Decomposition.

Images/videos captured from outdoor visual devices are usually degraded by turbid media, such as haze, smoke, fog, rain, and snow. Haze is the most common one in outdoor scenes due to the atmosphere conditions. In this paper, a novel deep learning-based architecture (denoted by MSRL-DehazeNet) for single image haze removal relying on multi-scale residual learning (MSRL) and image decomposition is proposed. Instead of learning an end-to-end mapping between each pair of hazy image and its corresponding haze-free one adopted by most existing learningbased approaches, we reformulate the problem as restoration of the image base component. Based on the decomposition of a hazy image into the base and the detail components, haze removal (or dehazing) can be achieved by both of our multi-scale deep residual learning and our simplified U-Net learning only for mapping between hazy and haze-free base components, while the detail component is further enhanced via the other learned convolutional neural network (CNN). Moreover, benefited by the basic building block of our deep residual CNN architecture and our simplified UNet structure, the feature maps (produced by extracting structural and statistical features), and each previous layer can be fully preserved and fed into the next layer. Therefore, possible color distortion in the recovered image would be avoided. As a result, the final haze-removed (or dehazed) image is obtained by integrating the haze-removed base and the enhanced detail image components. Experimental results have demonstrated good effectiveness of the proposed framework, compared with state-ofthe-art approaches.

Chromatic Titanium Photoanode for Dye-Sensitized Solar Cells under Rear Illumination.

Titanium (Ti) has high potential in many practical applications such as biomedicine, architecture, aviation, and energy. In this study, we demonstrate an innovative application of dye-sensitized solar cells (DSSCs) based on Ti photoanodes that can be integrated into the roof engineering of large-scale architectures. A chromatic Ti foil produced by anodizing oxidation (coloring) technology is an attractive roof material for large-scale architecture, showing a colorful appearance due to the formation of a reflective TiO2 thin layer on both surfaces of Ti. The DSSC is fabricated on the backside of the chromatic Ti foil using the Ti foil as the working electrode, and this roof-DSSC hybrid configuration can be designed as an energy harvesting device for indoor artificial lighting. Our results show that the facet-textured TiO2 layer on the chromatic Ti foil not only improves the optical reflectance for better light utilization but also effectively suppresses the charge recombination for better electron collection. The power conversion efficiency of the roof-DSSC hybrid system is improved by 30-40% with a main contribution from an improvement of short-circuit current density under standard 1 sun and dim-light (600-1000 lx) illumination.

Machine learning to relate PM2.5 and PM10 concentrations to outpatient visits for upper respiratory tract infections in Taiwan: A nationwide analysis.

AIM: To examine the accuracy of machine learning to relate particulate matter (PM) 2.5 and PM10 concentrations to upper respiratory tract infections (URIs). METHODS: Daily nationwide and regional outdoor PM2.5 and PM10 concentrations collected over 30 consecutive days obtained from the Taiwan Environment Protection Administration were the inputs for machine learning, using multilayer perceptron (MLP), to relate to the subsequent one-week outpatient visits for URIs. The URI data were obtained from the Centers for Disease Control datasets in Taiwan between 2009 and 2016. The testing used the middle month dataset of each season (January, April, July and October), and the training used the other months' datasets. The weekly URI cases were classified by tertile as high, moderate, and low volumes. RESULTS: Both PM concentrations and URI cases peak in winter and spring. In the nationwide data analysis, MLP machine learning can accurately relate the URI volumes of the elderly (89.05% and 88.32%, respectively) and the overall population (81.75% and 83.21%, respectively) with the PM2.5 and PM10 concentrations. In the regional data analyses, greater accuracy is found for PM2.5 than for PM10 for the elderly, particularly in the Central region (78.10% and 74.45%, respectively), whereas greater accuracy is found for PM10 than for PM2.5 for the overall population, particularly in the Northern region (73.19% and 63.04%, respectively). CONCLUSION: Short-term PM2.5 and PM10 concentrations were accurately related to the subsequent occurrence of URIs by using machine learning. Our findings suggested that the effects of PM2.5 and PM10 on URI may differ by age, and the mechanism needs further evaluation.

Propolin G, a prenylflavanone, isolated from Taiwanese propolis, induces caspase-dependent apoptosis in brain cancer cells.

We have previously shown that six propolins, A-F, could be isolated from Taiwanese propolis (TP) and that they exerted a broad spectrum of biological activities. Recently, we isolated a seventh compound, propolin G. Its chemical structure has been identified by NMR and fast atom bombardment-mass spectrometry spectra and was found to be identical to a known compound, nymphaeol C. We used high-performance liquid chromatography to determine the relative contents of propolins C, D, F, and G in TP collected in various seasons and regions and found them to be relatively higher in TPs collected from May to July than from September to October. In our present study, we were interested in the various biological activities of TP extract as well as in propolin G as a pure compound. We found that propolin G could efficiently induce apoptosis in brain cancer cell lines (glioma and glioblastoma). The apoptosis might have been through a mitochondrial- and caspase-dependent pathway. This result demonstrated that the TP collection season was more an important factor than the geographical region. Propolis has been suggested to possess a potent antioxidant activity. We further evaluated the antioxidant property of propolin G using DPPH (1,2-diphenyl-2-picryhydrazyl). Our results indicate that propolin G does possess free radical scavenging activity. We also evaluated the neuroprotective action of propolin G, TP, and BP (Brazilian propolis) extracts against oxidative stress in rat primary cortical neurons. Our data demonstrate that propolin G and TP extracts have a marked neuroprotective effect that is greater than BP extract. In conclusion, the isolation and characterization of propolin G from TP have demonstrated for the first time that this compound is a potent inducer of apoptosis in brain cancer cells and that this compound and TP extract exhibit a protective effect against oxidative stress in rat cortical neurons.

Enhanced B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation contributes to ABCC1-mediated chemoresistance and glutathione-mediated survival in acquired topoisomerase II poison-resistant cancer cells.

Nuclear factor erythroid-2-related factor 2 (NRF2) mainly regulates transcriptional activation through antioxidant-responsive elements (AREs) present in the promoters of NRF2 target genes. Recently, we found that NRF2 was overexpressed in a KB-derived drug-resistant cancer cell panel. In this panel, KB-7D cells, which show acquired resistance to topoisomerase II (Top II) poisons, exhibited the highest NRF2 activation. To investigate whether NRF2 directly contributed to acquired resistance against Top II poisons, we manipulated NRF2 by genetic and pharmacological approaches. The result demonstrated that silencing of NRF2 by RNA interference increased the sensitivity and treatment with NRF2 activator decreased the sensitivity of KB and KB-7D cells toward Top II poisons. Further, increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation activated NRF2 signaling in KB-7D cells. Moreover, increased binding of NRF2 to an ARE in the promoter of ATP-binding cassette subfamily C member 1 (ABCC1) directly contributed to Top II poison resistance. In addition, activation of NRF2 increased glutathione level and antioxidant capacity in KB-7D cells compared with that in KB cells; moreover, high glutathione level provided survival advantage to KB-7D cells. Our study is the first to show that aberrant NRF2 activation is via increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promote the survival of Top II poison-resistant cancer cells. Importantly, NRF2 downstream effectors ABCC1 and glutathione directly contribute to acquired resistance and survival, respectively. These results suggest that blockade of NRF2 signaling may enhance therapeutic efficacy and reduce the survival of Top II poison-refractory tumors in clinical.

Identification of an HptB-mediated multi-step phosphorelay in Pseudomonas aeruginosa PAO1.

We herein demonstrate that the hybrid sensor PA1611 carries out specific signal transduction, through HptB (PA3345), to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. As assessed by phenotypic changes in the hptB deletion mutant, the pathway is likely to be involved in the regulation of flagellar activity, the chemotaxis response, twitching motility, and biofilm formation in the bacteria.

Novel oxime-bearing coumarin derivatives act as potent Nrf2/ARE activators in vitro and in mouse model.

We have designed and synthesized certain novel oxime- and amide-bearing coumarin derivatives as nuclear factor erythroid 2 p45-related factor 2 (Nrf2) activators. The potency of these compounds was measured by antioxidant responsive element (ARE)-driven luciferase activity, level of Nrf2-related cytoprotective genes and proteins, and antioxidant activity. Among them, (Z)-3-(2-(hydroxyimino)-2-phenylethoxy)-2H-chromen-2-one (17a) was the most active, and more potent than the positive t-BHQ in the induction of ARE-driven luciferase activity. Exposure of HSC-3 cells to various concentrations of 17a strongly increased Nrf2 nuclear translocation and the expression level of Nrf2-mediated cytoprotective proteins in a concentration-dependent manner. HSC-3 cells pretreated with 17a significantly reduced t-BOOH-induced oxidative stress. In the animal experiment, Nrf2-mediated cytoprotective proteins, such as aldo-keto reductase 1 subunit C-1 (AKR1C1), glutathione reductase (GR), and heme oxygenase (HO-1), were obviously elevated in the liver of 17a-treated mice than that of control. These results suggested that novel oxime-bearing coumarin 17a is able to activate Nrf2/ARE pathway in vivo and are therefore seen as a promising candidate for further investigation.

EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.

Misfolded proteins of the endoplasmic reticulum (ER) are eliminated by the ER-associated degradation (ERAD) in eukaryotes. In S. cerevisiae, ER-resident lectins mediate substrate recognition through bipartite signals consisting of an unfolded local structure and the adjacent glycan. Trimming of the glycan is essential for the directional delivery of the substrates. Whether a similar recognition and delivery mechanism exists in mammalian cells is unknown. In this study, we systematically study the function and substrate specificity of known mammalian ER lectins, including EDEM1/2/3, OS-9 and XTP-3B using the recently identified ERAD substrate sonic hedgehog (SHH), a soluble protein carrying a single N-glycan, as well as its nonglycosylated mutant N278A. Efficient ERAD of N278A requires the core processing complex of HRD1, SEL1L and p97, similar to the glycosylated SHH. While EDEM2 was required for ERAD of both glycosylated and non-glycosylated SHHs, EDEM3 was only necessary for glycosylated SHH and EDEM1 was dispensable for both. Degradation of SHH and N278A also required OS-9, but not the related lectin XTP3-B. Robust interaction of both EDEM2 and OS-9 with a non-glycosylated SHH variant indicates that the misfolded polypeptide backbone, rather than a glycan signature, functions as the predominant signal for recognition for ERAD. Notably, SHH-N278A is the first nonglycosylated substrate to require EDEM2 for recognition and targeting for ERAD. EDEM2 also interacts with calnexin and SEL1L, suggesting a potential avenue by which misfolded glycoproteins may be shunted towards SEL1L and ERAD rather than being released into the secretory pathway. Thus, ER lectins participate in the recognition and delivery of misfolded ER substrates differently in mammals, with an underlying mechanism distinct from that of S. cerevisiae.

Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.

The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly.