Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

A Bifunctional Role for the UHRF1 UBL Domain in the Control of Hemi-methylated DNA-Dependent Histone Ubiquitylation.

DNA methylation patterns regulate gene expression programs and are maintained through a highly coordinated process orchestrated by the RING E3 ubiquitin ligase UHRF1. UHRF1 controls DNA methylation inheritance by reading epigenetic modifications to histones and DNA to activate histone H3 ubiquitylation. Here, we find that all five domains of UHRF1, including the previously uncharacterized ubiquitin-like domain (UBL), cooperate for hemi-methylated DNA-dependent H3 ubiquitin ligation. Our structural and biochemical studies, including mutations found in cancer genomes, reveal a bifunctional requirement for the UBL in histone modification: (1) the UBL makes an essential interaction with the backside of the E2 and (2) the UBL coordinates with other UHRF1 domains that recognize epigenetic marks on DNA and histone H3 to direct ubiquitin to H3. Finally, we show UBLs from other E3s also have a conserved interaction with the E2, Ube2D, highlighting a potential prevalence of interactions between UBLs and E2s.

Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

Inside-Out Design of Zinc-Binding Proteins with Non-Native Backbones.

The de novo design of functional proteins requires specification of tertiary structure and incorporation of molecular binding sites. Here, we develop an inside-out design strategy in the molecular modeling program Rosetta that begins with amino acid side chains from one or two α-helices making well-defined contacts with a ligand. A full-sized protein is then built around the ligand by adding additional helices that promote the formation of a protein core and allow additional contacts with the ligand. The protocol was tested by designing 12 zinc-binding proteins, each with 4-5 helices. Four of the designs were folded and bound to zinc with equilibrium dissociation constants varying between 95 nM and 1.1 µM. The design with the tightest affinity for zinc, N12, adopts a unique conformation in the folded state as assessed with nuclear magnetic resonance (NMR) and the design model closely matches (backbone root-mean-square deviation (RMSD) < 1 Å) an AlphaFold model of the sequence. Retrospective analysis with AlphaFold suggests that the sequences of many of the failed designs did not encode the desired tertiary packing.

Insight into Viral Hijacking of CRL4 Ubiquitin Ligase through Structural Analysis of the pUL145-DDB1 Complex.

Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway-regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. Members of the Cullin-RING family of ubiquitin (Ub) ligases are frequently co-opted by divergent viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING (CRL4)-DDB1 Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including the V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of human cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these divergent viral sequences hijack DDB1 is not well understood. Here, we use a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs appear to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and also acquire favorable interactions at neighboring residues within the H-box. Overall, these studies reveal how viruses evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery. IMPORTANCE Many different viruses modulate the protein machinery required for ubiquitination to enhance viral fitness. Specifically, several viruses hijack the cullin-RING ligase CRL4DDB1 to degrade host resistance factors. Human cytomegalovirus (HCMV) encodes pUL145 that redirects CRL4DDB1 to evade the immune system through the targeted degradation of the antiviral immune response protein STAT2. However, it is unclear why several viruses bind specific surfaces on ubiquitin ligases to repurpose their activity. We demonstrate that viruses have optimized H-box motifs that bind DDB1 with higher affinity than the H-box of native binders. For viral H-boxes, native interactions are maintained, but additional interactions that are absent in host cell H-boxes are formed, indicating that rewiring CRL4DDB1 creates a selective advantage for the virus. The DDB1-pUL145 peptide structure reveals that water-mediated interactions are critical to the higher affinity. Together, our data present an interesting example of how viral evolution can exploit a weakness in the ubiquitination machinery.

Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler.

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

The effect of the bgs13 mutation on the structure of the reporter protein beta-lactoglobulin: Influence on folding and aggregation in Pichia pastoris.

Pichia pastoris, a methylotrophic yeast used for recombinant protein expression, has the capability of performing many eukaryotic post-translational modifications, growing to high cell densities, and producing proteins in a cost-effective manner. However, P. pastoris's secretion properties are not always efficient, and its secretory pathway mechanisms have not been thoroughly elucidated. A previously identified mutant strain, bgs13, was found to efficiently secrete most recombinant proteins tested, raising the possibility that this bgs13 mutant is a universal super secreter. In this study, we used a reporter protein, ß-lactoglobulin (b-LG), to perform structural analysis of the protein secreted from wild type and mutant bgs13 strains to investigate the secretory mechanism. Primary, secondary, and tertiary structures of b-LG were examined using Edman sequencing, circular dichroism, tryptophan fluorescence, and temperature induced aggregation analysis. Our results demonstrate that the bgs13 produced more b-LG than the wt strain and that this protein was functionally folded similar to the wt. Surprisingly, we also found that the bgs13 b-LG was more resistant to aggregation, providing another example of the superior qualities of this strain for enhanced secreted protein production.

Enhancement of cell proliferation and motility of mammalian cells grown in co-culture with Pichia pastoris expressing recombinant human FGF-2.

Many studies examining the biological function of recombinant proteins and their effects on the physiology of mammalian cells stipulate that the proteins be purified before being used as therapeutic agents. In this study, we explored the possibility of using unpurified recombinant proteins to treat mammalian cells. The recombinant protein was used directly from the expression source and the biological function was compared to purified commercially available, equivalent protein. The model for this purpose was recombinant FGF-2, expressed by Pichia pastoris, which was used to treat the murine fibroblast cell line, NIH/3T3. We generated a P. pastoris strain (yHL11) that constitutively secreted a biologically active recombinant FGF-2 protein containing an N-terminal c-myc epitope (Myc-FGF-2). Myc-FGF-2 was then used without purification either a) in the form of conditioned mammalian cell culture medium or b) during co-cultures of yHL11 with NIH/3T3 to induce higher proliferation and motility of NIH/3T3 cells. The effects of Myc-FGF-2 on cell physiology were comparable to commercially available FGF-2. To our knowledge, this is the first time the physiology of cultured mammalian cells had been successfully altered with a recombinant protein secreted by P. pastoris while the two species shared the same medium and culture conditions. Our data demonstrated the biological activity of unpurified recombinant FGF-2 on NIH/3T3 cells and provided a foundation for directly using unpurified recombinant proteins expressed by P. pastoris with mammalian cells, potentially as wound-healing therapeutics.

Comparative biochemical analysis of UHRF proteins reveals molecular mechanisms that uncouple UHRF2 from DNA methylation maintenance.

UHRF1 is a histone- and DNA-binding E3 ubiquitin ligase that functions with DNMT1 to maintain mammalian DNA methylation. UHRF1 facilitates DNMT1 recruitment to replicating chromatin through a coordinated mechanism involving histone and DNA recognition and histone ubiquitination. UHRF2 shares structural homology with UHRF1, but surprisingly lacks functional redundancy to facilitate DNA methylation maintenance. Molecular mechanisms uncoupling UHRF2 from DNA methylation maintenance are poorly defined. Through comprehensive and comparative biochemical analysis of recombinant human UHRF1 and UHRF2 reader and writer activities, we reveal conserved modes of histone PTM recognition but divergent DNA binding properties. While UHRF1 and UHRF2 diverge in their affinities toward hemi-methylated DNA, we surprisingly show that both hemi-methylated and hemi-hydroxymethylated DNA oligonucleotides stimulate UHRF2 ubiquitin ligase activity toward histone H3 peptide substrates. This is the first example of an E3 ligase allosterically regulated by DNA hydroxymethylation. However, UHRF2 is not a productive histone E3 ligase toward purified mononucleosomes, suggesting UHRF2 has an intra-domain architecture distinct from UHRF1 that is conformationally constrained when bound to chromatin. Collectively, our studies reveal that uncoupling of UHRF2 from the DNA methylation maintenance program is linked to differences in the molecular readout of chromatin signatures that connect UHRF1 to ubiquitination of histone H3.

Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate.

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

The nature and consequences of cognitive deficits among tobacco smokers with HIV: a comparison to tobacco smokers without HIV.

HIV-infected smokers lose more years of life to tobacco-related disease than HIV. Since neurocognitive deficits are common among those with HIV and are associated with smoking persistence, these deficits may be a unique barrier to smoking cessation among HIV-infected smokers. Documenting unique differences in and correlates of cognition among HIV-infected smokers is a critical step towards developing a population-specific tobacco cessation treatment. We compared neurocognitive function between HIV-infected (n = 103) and HIV-uninfected smokers (n = 70), accounting for demographic and smoking-related variables. We also evaluated whether HIV-related health outcomes (e.g., CD4 count, viral load, depression ratings, quality of life [QoL]) and HAART adherence were associated with cognition. Participants completed neurocognitive tasks (N-back and Continuous Performance Task [CPT]) measuring working memory, attention, and processing speed, and intra-individual variability. Stepwise regression models were conducted and validated with resampling techniques. HIV-infected smokers performed worse than HIV-uninfected smokers on working memory, processing speed, and intra-individual variability (all p < 0.01). ROC analysis for the model including cognitive measures demonstrated 85% area under the curve, which indicates "good prediction" for distinguishing between HIV-infected and HIV-uninfected smokers. This was a significant improvement over the model including demographic and smoking-related variables only (p = 0.0003). Among HIV-infected smokers, neurocognitive performance was negatively associated with QoL and depression ratings. Smoking cessation interventions for HIV-infected smokers should consider cognitive neurorehabilitation as a potential strategy to decrease the likelihood of nicotine relapse and decrease tobacco-related morbidity in this population.

Continuous Abstinence During Early Alcohol Treatment is Significantly Associated with Positive Treatment Outcomes, Independent of Duration of Abstinence.

AIMS: Neither the predictive value of early continuous abstinence in alcohol use disorder (AUD) or the point at which this effect may emerge has been evaluated. This analysis of the Combined Pharmacotherapies and Behavioral Interventions (COMBINE) clinical trial evaluated whether abstinence early in treatment was a predictor of longer term abstinence. METHODS: Participants who stated a goal of total abstinence (N = 954) were dichotomized into Early Abstainer vs. Nonabstainers and were compared on a variety of drinking outcome measures that are frequently used in clinical trial evaluations of alcohol treatment strategies, as a function of duration of early continuous abstinence. RESULTS: Significant differences existed for every outcome. Early Abstinence was significantly associated with fewer drinks per drinking day, number of drinking and number of heavy drinking days, and longer time to first drinking and first heavy drinking day. Effects were evident within the first week. The magnitude of all effects increased as the duration of early abstinence (1-4 weeks) increased, though the size of increase varied across the outcomes. CONCLUSIONS: These data provide evidence that drinking at the beginning of alcohol treatment is significantly and robustly associated with drinking throughout and at the end of a clinical trial treatment for AUD. Early drinking may be a useful early index to identify whether patients are responding positively to a treatment strategy, and provides a useful method for tailoring treatment to patients that is consistent with a personalized medicine approach.

Assessing the extent and nature of wildlife trade on the dark web.

Use of the internet as a trade platform has resulted in a shift in the illegal wildlife trade. Increased scrutiny of illegal wildlife trade has led to concerns that online trade of wildlife will move onto the dark web. To provide a baseline of illegal wildlife trade on the dark web, we downloaded and archived 9852 items (individual posts) from the dark web, then searched these based on a list of 121 keywords associated with illegal online wildlife trade, including 30 keywords associated with illegally traded elephant ivory on the surface web. Results were compared with items known to be illegally traded on the dark web, specifically cannabis, cocaine, and heroin, to compare the extent of the trade. Of these 121 keywords, 4 resulted in hits, of which only one was potentially linked to illegal wildlife trade. This sole case was the sale and discussion of Echinopsis pachanoi (San Pedro cactus), which has hallucinogenic properties. This negligible level of activity related to the illegal trade of wildlife on the dark web relative to the open and increasing trade on the surface web may indicate a lack of successful enforcement against illegal wildlife trade on the surface web.

Estimating the extent and structure of trade in horticultural orchids via social media.

The wildlife trade is a lucrative industry involving thousands of animal and plant species. The increasing use of the internet for both legal and illegal wildlife trade is well documented, but there is evidence that trade may be emerging on new online technologies such as social media. Using the orchid trade as a case study, we conducted the first systematic survey of wildlife trade on an international social-media website. We focused on themed forums (groups), where people with similar interests can interact by uploading images or text (posts) that are visible to other group members. We used social-network analysis to examine the ties between 150 of these orchid-themed groups to determine the structure of the network. We found 4 communities of closely linked groups based around shared language. Most trade occurred in a community that consisted of English-speaking and Southeast Asian groups. In addition to the network analysis, we randomly sampled 30 groups from the whole network to assess the prevalence of trade in cultivated and wild plants. Of 55,805 posts recorded over 12 weeks, 8.9% contained plants for sale, and 22-46% of these posts pertained to wild-collected orchids. Although total numbers of posts about trade were relatively small, the large proportion of posts advertising wild orchids for sale supports calls for better monitoring of social media for trade in wild-collected plants.

Reduction of volatile fatty acids and odor offensiveness by anaerobic digestion and solid separation of dairy manure during manure storage.

Volatile fatty acids (VFA) play an important role in the biodegradation of organic wastes and production of bioenergy under anaerobic digestion, and are related to malodors. However, little is known about the dynamics of VFA during dairy manure storage. This study evaluated the characteristics of VFA in dairy manure before and after anaerobic co-digestion in a laboratory experiment using eight lab-scale reactors. The reactors were loaded with four different types of dairy manure: (1) liquid dairy manure from a freestall barn, (2) mixture of dairy manure and co-digestion food processing wastes at the inlet of an anaerobic digester, (3) effluent from the digester outlet, and (4) the liquid fraction of effluent from a solid separator. Four VFA (acetic, propionic, butyric, and 2-methylbutyric acids) were identified and quantified in weekly manure samples from all reactors. Results showed that the dominant VFA was acetic acid in all four manure sources. The off-farm co-digestion wastes significantly increased the total VFA concentrations and the proportions of individual VFA in the influent. The dairy manure under storage demonstrated high temporal and spatial variations in pH and VFA concentrations. Anaerobic digestion reduced the total VFA by 86%-96%; but solid-liquid separation did not demonstrate a significant reduction in total VFA in this study. Using VFA as an indicator, anaerobic digestion exhibited an effective reduction of dairy manure odor offensiveness.

Recruitment techniques for alcohol pharmacotherapy clinical trials: A cost-benefit analysis.

OBJECTIVES: Alcohol use disorders (AUDs) represent a large public health burden with relatively few efficacious pharmacotherapies. Randomized controlled trials (RCTs) for new AUD therapies can be hampered by ineffective recruitment, leading to increased trial costs. The current analyses examined the effectiveness of recruitment efforts during two consecutive outpatient RCTs of novel AUD pharmacotherapies conducted between 2009 and 2012. METHODS: During an initial phone screen, participants identified an ad source for learning about the study. Qualified persons were then scheduled for in-person screens. The present analyses examined demographic differences amongst the eight ad sources utilized. Recruitment effectiveness was determined by dividing the number of persons meeting criteria for an in-person screen by the total number of callers from each ad source. Cost-effectiveness was determined by dividing total ad source cost by number of screens, participants randomized, and completers. RESULTS: 1,813 calls resulted in 1,005 completed phone screens. The most common ad source was TV (34%), followed by print (29%), word-of-mouth (11%), flyer (8%), internet (5%), radio (5%), bus ad (2%), and billboard (1%). Participants reporting bus ads (46%), billboard (44%), or print ads (34%) were significantly more likely than the other sources to meet criteria to be scheduled for in-person screens. The most cost-effective ad source was print ($2,506 per completer), while bus ad was the least cost-effective ($13,376 per completer). CONCLUSIONS: Recruitment in AUD RCTs can be successful using diverse advertising methods. The present analyses favored use of print ads as most cost-effective.

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.