Hypomorphic variants in the SEL1L-HRD1 ER-associated degradation (ERAD) complex have been linked to severe neurological syndromes in children, including neurodevelopmental delay, intellectual disability, motor dysfunction, and early death. Despite this association, its physiological importance and underlying mechanisms in neurons remain poorly understood. Here, we show that neuronal SEL1L-HRD1 ERAD is essential for maintaining one-carbon metabolism, motor function, and overall viability. Neuron-specific deletion of Sel1L in mice (Sel1LSynCre) resulted in growth retardation, severe motor impairments, and early mortality by 9 weeks of age - mirroring core clinical features observed in affected patients - despite preserved neuronal numbers and only modest ER stress. Multiomics analyses, including single-nucleus RNA sequencing and metabolomics, revealed significant dysregulation of one-carbon metabolism in ERAD-deficient brains. This included activation of the serine, folate, and methionine pathways, accompanied by elevated levels of S-adenosylmethionine and related metabolites, likely resulting from induction of the integrated stress response. Together, these findings uncover a previously unappreciated role for neuronal SEL1L-HRD1 ERAD in coordinating ER protein quality control with metabolic adaptation, providing insight into the molecular basis of ERAD-related neurodevelopmental disease.

Hypomorphic variants in the SEL1L-HRD1 ER-associated degradation (ERAD) complex have been linked to severe neurological syndromes in children, including neurodevelopmental delay, intellectual disability, motor dysfunction, and early death. Despite this association, its physiological importance and underlying mechanisms in neurons remain poorly understood. Here, we show that neuronal SEL1L-HRD1 ERAD is essential for maintaining one-carbon metabolism, motor function, and overall viability. Neuron-specific deletion of Sel1L in mice (Sel1L SynCre ) resulted in growth retardation, severe motor impairments, and early mortality by 9 weeks of age-mirroring core clinical features observed in affected patients-despite preserved neuronal numbers and only modest ER stress. Multi-omics analyses, including single-nucleus RNA sequencing and metabolomics, revealed significant dysregulation of one-carbon metabolism in ERAD-deficient brains. This included activation of the serine, folate, and methionine pathways, accompanied by elevated levels of S-adenosylmethionine and related metabolites, likely resulted from induction of the integrated stress response (ISR). Together, these findings uncover a previously unappreciated role for neuronal SEL1L-HRD1 ERAD in coordinating ER protein quality control with metabolic adaptation, providing new insight into the molecular basis of ERAD-related neurodevelopmental disease.

Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae , we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the endoplasmic reticulum associated degradation (ERAD) system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly conserved ERAD system.

Over one-third of all proteins pass through the endoplasmic reticulum (ER) and must undergo folding events to reach their native state. Protein folding is an error-prone process, and misfolded proteins can be removed from the ER lumen and membrane by ER-associated degradation (ERAD). Hrd1 is a ubiquitin ligase that is a central component in ERAD of lumenal and transmembrane proteins in all eukaryotes. However, the sequences allowing recognition by Hrd1 (called degrons) are largely unidentified. Degrons are commonly short, linear sequences of amino acids that are sufficient for targeting proteins for degradation. We have identified the first groups of degrons that function within the ER through Hrd1-mediated ERAD, followed by degradation by the cytosolic proteasome. These degrons were identified through screening penta-amino acid libraries in S. cerevisiae and validated in both S. cerevisiae and mammalian cell lines. We demonstrate that the N-terminal degrons function for both soluble lumenal and integral ER membrane proteins and provide useful tools for driving degradation from the ER lumen. J.D. was supported by the NIH Pharmacological Sciences Training Grant (T32-GM140223). R.S. (previously R. Plumb) was supported by an NIH/NIGMS Award (5F32GM136020) and D.D. was supported by the NIH Cellular and Molecular Biology Training Grant (T32-GM007315). In addition, this work was supported by an NIH/NIGMS Award (R35GM128592 to R.D.B.).

Misfolded endoplasmic reticulum proteins are degraded through a process called endoplasmic reticulum associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identified several regions required for different Hrd1 functions. Most surprisingly, we found two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we defined roles for disordered regions between structural elements that were required for Hrd1's ability to autoubiquitinate and interact with substrate. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the endoplasmic reticulum membrane.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as ER-associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, and ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition influences ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long-chain ceramides leads to a marked increase in the level of ubiquitinated substrates in the ER membrane and concomitantly reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized mechanism in which ER membrane lipid remodeling changes the activity of ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as endoplasmic reticulum associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition controls ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long chain ceramides leads to a dramatic increase in the level of ubiquitinated substrates in the ER membrane, and concomitantly, reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized regulatory mechanism in which ER membrane lipid remodeling controls the ERAD system.

Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. Whereas much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is un-known. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFα subunit of these heterodimeric transcription factors comparable with hypoxia with activation of downstream target genes. The addition of fatty acids to culture medium suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.

Hypoxia‐inducible factor (HIF) transcription factors are central regulators of oxygen homeostasis, controlling energy utilization, erythrogenesis, angiogenesis and cancer metabolism. Oxygen negatively regulates HIF activity by promoting HIFα prolyl hydroxylation and proteasome‐dependent degradation. Using human pancreatic adenocarcinoma cells, we demonstrate that serum lipoproteins are an independent regulator of HIF under normoxia. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of HIFα comparable to hypoxia with activation of downstream target genes. Addition of unsaturated fatty acids suppressed this signal, which required an intact mitochondrial respiratory chain. A survey of different cell lines showed that this signaling pathway functions in a wide range of tissues. These studies show that lipoprotein and oxygen supply are distinct signals integrated to control HIF activity and identify serum lipids as a potential modulator of HIF pathway activity.Support or Funding InformationNational Institutes of Health, HL077588 and GM126088This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endoplasmic reticulum (ER)-associated degradation (ERAD) and the unfolded protein response (UPR) are two key quality-control machineries in the cell. ERAD is responsible for the clearance of misfolded proteins in the ER for cytosolic proteasomal degradation, while UPR is activated in response to the accumulation of misfolded proteins. It has long been thought that ERAD is an integral part of UPR because expression of many ERAD genes is controlled by UPR; however, recent studies have suggested that ERAD has a direct role in controlling the protein turnover and abundance of IRE1 alpha, the most conserved UPR sensor. Here, we review recent advances in our understanding of IRE1 alpha activation and propose that UPR and ERAD engage in an intimate crosstalk to define folding capacity and maintain homeostasis in the ER.

Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

Hypoxic growth of fungi requires sterol regulatory element-binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane-anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi-localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA-ATPase Cdc48 through a C-terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48-binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation.

The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein-protein interactions. APEX2 fused to a protein of interest covalently tags nearby proteins with biotin-phenol (BP) when H2O2 is added to the cell culture medium. Subsequent affinity purification of biotinylated proteins allows for identification by MS. BP labelling occurs in 1 min, providing temporal control of labelling. The APEX2 tool enables proteomic mapping of subcellular compartments as well as identification of dynamic protein complexes, and has emerged as a new methodology for proteomic analysis. Despite these advantages, a related APEX2 approach has not been developed for yeast. Here we report methods to enable APEX2-mediated biotin labelling in yeast. Our work demonstrated that high osmolarity and disruption of cell wall integrity permits live-cell biotin labelling in Schizosaccharomyces pombe and Saccharomyces cerevisiae respectively. Under these conditions, APEX2 permitted targeted and proximity-dependent labelling of proteins. The methods described herein set the stage for large-scale proteomic studies in yeast. With modifications, the method is also expected to be effective in other organisms with cell walls, such as bacteria and plants.

Proteasomes generally degrade substrates tagged with polyubiquitin chains. In rare cases, however, proteasomes can degrade proteins without prior ubiquitination. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasome-dependent, ubiquitin-independent degradation of the retinoblastoma (Rb) and Daxx proteins. These transcriptional corepressors and tumor suppressors inhibit the expression of cellular or viral genes that are required for efficient viral replication. Proteasomes are composed of a 20S catalytic core with or without one or two activator complexes, of which there are four different types. Here, we show that only one of these activators, the 19S regulatory particle that normally participates in ubiquitin-dependent protein degradation, is required for pp71-mediated degradation of Rb and Daxx. We report the unique use of a well-established route of substrate delivery to the proteasome by a viral protein to promote infection.

ABSTRACT Elongins B and C are members of complexes that increase the efficiency of transcriptional elongation by RNA polymerase II (RNAPII) and enhance the monoubiquitination of histone H2B, an epigenetic mark of actively transcribed genes. Here we show that, in addition to its role in facilitating transcription of the cellular genome, elongin B also enhances gene expression from the double-stranded DNA genome of human cytomegalovirus (HCMV), a pathogenic herpesvirus. Reducing the level of elongin B by small interfering RNA- or short hairpin RNA-mediated knockdown decreased viral mRNA expression, viral protein accumulation, viral DNA replication, and infectious virion production. Chromatin immunoprecipitation analysis indicated viral genome occupancy of the elongating form of RNAPII, and monoubiquitinated histone H2B was reduced in elongin B-deficient cells. These data suggest that, in addition to the previously documented epigenetic regulation of transcriptional initiation, HCMV also subverts cellular elongin B-mediated epigenetic mechanisms for enhancing transcriptional elongation to enhance viral gene expression and virus replication. IMPORTANCE The genetic and epigenetic control of transcription initiation at both cellular and viral promoters is well documented. Recently, the epigenetic modification of histone H2B monoubiquitination throughout the bodies of cellular genes has been shown to enhance the elongation of RNA polymerase II-initiated transcripts. Mechanisms that might control the elongation of viral transcripts are less well studied. Here we show that, as with cellular genes, elongin B-mediated monoubiquitination of histone H2B also facilitates the transcriptional elongation of human cytomegalovirus genes. This and perhaps other epigenetic markings of actively transcribed regions may help in identifying viral genes expressed during in vitro latency or during natural infections of humans. Furthermore, this work identifies a novel, tractable model system to further study the regulation of transcriptional elongation in living cells.

Most eukaryotic proteins destined for imminent destruction are first tagged with a chain of ubiquitin molecules and are subsequently dismantled by the proteasome. Ubiquitin-independent degradation of substrates by the proteasome, however, also occurs. The number of documented proteasome-dependent, ubiquitin-independent degradation events remains relatively small but continues to grow. Proteins involved in oncogenesis and tumor suppression make up the majority of the known cases for this type of protein destruction. Provocatively, viruses with confirmed or suspected oncogenic properties are also prominent participants in the pantheon of ubiquitin-independent proteasomal degradation events. In this review, we identify and describe examples of proteasome-dependent, ubiquitin-independent protein degradation that occur during tumor virus infections, speculate why this type of protein destruction may be preferred during oncogenesis, and argue that this uncommon type of protein turnover represents a prime target for antiviral and anticancer therapeutics.

Small ubiquitin-like modifiers (SUMOs) are covalently conjugated to target proteins to regulate numerous biological processes, including subcellular localization, protein–protein interactions, and transactivational activities. While the majority of identified SUMO targets are cellular proteins, SUMO modified viral proteins have also been identified. In addition, there are a growing number of examples where viruses alter the sumoylation status of host cell proteins. Work from our laboratory has previously demonstrated that the human cytomegalovirus (HCMV) virion tegument protein pp71 binds to Daxx, a cellular transcriptional co-repressor, and promotes its sumoylation. Here we describe the in vivo techniques used to detect pp71-induced sumoylation of Daxx in a cotransfection system as well as the endogenous SUMO modified form of Daxx in HCMV-infected cells. The approaches we describe can be easily adapted to infections with other viruses and for the detection of sumoylation of other proteins.

Proteins that participate in a diverse array of cellular processes can be modified covalently and reversibly on lysine residues by the small ubiquitin-like modifier proteins termed SUMOs. In some instances, such modification profoundly affects protein function, but the biological significance of many SUMOylation events remains unknown. Protein SUMOylation is modulated during many viral infections. Here we demonstrate that the human cytomegalovirus (HCMV) pp71 protein promotes the SUMOylation of its cellular substrate, Daxx. A component of promyelocytic leukemia nuclear bodies, Daxx is a transcriptional corepressor that silences the expression of viral immediate-early (IE) genes at the start of both lytic and quiescent HCMV infections. pp71 is a tegument component delivered directly to cells by infecting HCMV virions. At the start of lytic infections, it travels to the nucleus and stimulates viral IE gene expression by displacing the chromatin remodeling protein ATRX from Daxx and by mediating Daxx degradation through a rare ubiquitin-independent, proteasome-dependent process. Here we report that pp71 also substantially increases the basal level of SUMOylated Daxx observed in cells. To date, consequences of Daxx SUMOylation have not been observed for cellular promoters, and we detected no qualitative change in viral IE gene expression in the absence of pp71-induced Daxx SUMOylation. Thus, while pp71 enhances the basal level of SUMOylated Daxx, the role that this modification plays in regulating Daxx activity in uninfected or HCMV-infected cells remains an enigma.

ABSTRACT Proteins that participate in a diverse array of cellular processes can be modified covalently and reversibly on lysine residues by the small ubiquitin-like modifier proteins termed SUMOs. In some instances, such modification profoundly affects protein function, but the biological significance of many SUMOylation events remains unknown. Protein SUMOylation is modulated during many viral infections. Here we demonstrate that the human cytomegalovirus (HCMV) pp71 protein promotes the SUMOylation of its cellular substrate, Daxx. A component of promyelocytic leukemia nuclear bodies, Daxx is a transcriptional corepressor that silences the expression of viral immediate-early (IE) genes at the start of both lytic and quiescent HCMV infections. pp71 is a tegument component delivered directly to cells by infecting HCMV virions. At the start of lytic infections, it travels to the nucleus and stimulates viral IE gene expression by displacing the chromatin remodeling protein ATRX from Daxx and by mediating Daxx degradation through a rare ubiquitin-independent, proteasome-dependent process. Here we report that pp71 also substantially increases the basal level of SUMOylated Daxx observed in cells. To date, consequences of Daxx SUMOylation have not been observed for cellular promoters, and we detected no qualitative change in viral IE gene expression in the absence of pp71-induced Daxx SUMOylation. Thus, while pp71 enhances the basal level of SUMOylated Daxx, the role that this modification plays in regulating Daxx activity in uninfected or HCMV-infected cells remains an enigma.

The cellular Daxx protein represses human cytomegalovirus (HCMV) gene expression from the major immediate early promoter. HCMV prevents Daxx-mediated silencing during lytic infection by delivering the viral pp71 tegument protein to the nucleus, where pp71 binds to and induces the proteasomal degradation of Daxx. In this study, we show that a functional ubiquitin pathway is not required for the proteasomal degradation of the endogenous Daxx protein by tegument-delivered pp71 in HCMV-infected cells, demonstrating that the pp71-mediated degradation of Daxx occurs through a proteasome-dependent, ubiquitin-independent pathway.