Single E2 Research Articles

Phage defence system CBASS is regulated by a prokaryotic E2 enzyme that imitates the ubiquitin pathway.

The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.

Structural insight into N-degron polyubiquitination catalyzed by Ubr1

The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches.

Structural insights into Ubr1-mediated N-degron polyubiquitination.

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.

A modified semi-weight function method for stress intensity factor calculation of a vertical crack terminating at the interface

The interfacial crack stress intensity factor (SIF) calculation plays an important role in the safety evaluation and anti-crack design for multilayer structures. The current analytical or numerical methods have difficulty of complicated auxiliary functions or special crack-tip singular elements. In this study, a modified semi-weight function method was proposed based on the submatrix method to calculate the SIFs of the crack vertical to and terminating at the interface of bimaterial (easily appears in compressed air energy storage) and verified by two calculation examples compared with the available literature results. The effects of bimaterial parameters (Ei, υi), relative crack length (a/w2) and outer integration path (r2) on the SIFs were also analyzed. Research results show that the SIFs (K1 and K2) are related to only the ratio of E2/E1 independent of the single E1 or E2, and they are increased with increase of a/w2. The reasonable range of r2 is rs < r2 < a/2 (rs is minimum finite element size). The modified semi-weight function method has advantages of unified auxiliary function for different crack sizes and external loads and simpler calculation process without any crack-tip singular elements over current methods.

Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways.

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

Preliminary Validation of Electroporation-Electrolysis (E2) for Cardiac Ablation Using a Parameterisable In-Vivo Model.

Atrial fibrillation is the most common arrhythmia, increasing the risk of stroke, heart failure and death, and a growing epidemic. Electroporation ablation is emerging in cardiac ablation for atrial fibrillation as a fast, tissue-specific and non-thermal alternative to existing technologies tied by their thermal action to shortcomings in efficacy, speed and risk. Studies so far have aimed to translate the success of irreversible electroporation from tumour treatment, with its kilovolt pulses, to cardiac ablation. However, these high voltages may be less appealing for cardiac ablation from clinical, technical and regulatory standpoints. A novel ablation technique combining electroporation and electrolysis in a single pulse E2 uses lower voltages. A custom E2 ablation system was developed and tested on an in vivo tissue model. Histopathological analysis showed lesions of clinically relevant depth, achieved without any acute complications or severe muscle contractions. Lesions were mapped onto a numerical model developed to refine further prototyping. This study provides preliminary prototype validation and the methodological foundation for dose optimisation towards endocardial application.

The STUbL RNF4 regulates protein group SUMOylation by targeting the SUMO conjugation machinery

SUMO-targeted ubiquitin ligases (STUbLs) mediate the ubiquitylation of SUMOylated proteins to modulate their functions. In search of direct targets for the STUbL RNF4, we have developed TULIP (targets for ubiquitin ligases identified by proteomics) to covalently trap targets for ubiquitin E3 ligases. TULIP methodology could be widely employed to delineate E3 substrate wiring. Here we report that the single SUMO E2 Ubc9 and the SUMO E3 ligases PIAS1, PIAS2, PIAS3, ZNF451, and NSMCE2 are direct RNF4 targets. We confirm PIAS1 as a key RNF4 substrate. Furthermore, we establish the ubiquitin E3 ligase BARD1, a tumor suppressor and partner of BRCA1, as an indirect RNF4 target, regulated by PIAS1. Interestingly, accumulation of BARD1 at local sites of DNA damage increases upon knockdown of RNF4. Combined, we provide an insight into the role of the STUbL RNF4 to balance the role of SUMO signaling by directly targeting Ubc9 and SUMO E3 ligases.

Green microalgae in removal and biotransformation of estradiol and ethinylestradiol

Estrogens, a group of steroid hormones, are serious environmental pollutants due to their persistence, ubiquity, and endocrine-disrupting toxicity. Natural and synthetic estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), respectively, have been found in the influent of municipal wastewater treatment plants. The removal and biotransformation of mixed or single E2 and EE2 by six microalgal species, including three local isolates of Chlamydomonas sp. (WW), Chlorella sp. (2f5aia), and Chlorella sp. (1uoai) and three commercially available species, namely Scenedesmus quadricauda (= Desmodesmus communis) (SQ), Selenastrum capricornutum (SC), and Chlorella vulgaris (CV), were compared. The biological removal of estrogen was incubation time- and species-dependent. Among six species, SC had the highest removal ability for both E2 and EE2; their removal increased with incubation time and achieved 93.9 and 75.3 % removal, respectively, at the end of incubation. After 7-day incubation, the removal percentage of mixed EE2 by SC was significantly higher than that of single EE2 (P ≤ 0.05). Differences in the removal percentages of E2 and EE2 between commercial and local species were not significant, as reflected by the comparable removal efficiency among three Chlorella species, CV, 1uoai, and 2f5aia, with the highest removal of E2, either in mixed or in single treatment, at day 2. In all species, the percentages of E2 or EE2 taken up into cells were very low (0.2–9.6 %) when compared to the respective biological removal percentages. E2 was easily transformed into estrone (E1) and some unknown products by all microalgal species, while the products for EE2 transformation could not be identified due to the limited availability of the standards for metabolites.

Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed "ER-associated degradation" (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed "Der3") and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.

Classification and interaction modes of 40 rice E2 ubiquitin-conjugating enzymes with 17 rice ARM-U-box E3 ubiquitin ligases

Rice, a monocot model crop, contains at least 48 putative E2 ubiquitin (Ub)-conjugating enzymes. Based on homology comparisons with 40 Arabidopsis E2 proteins and 35 human E2s, 48 rice E2s were classified into 15 different groups. Yeast two-hybrid analyses using the U-box-domain regions of armadillo (ARM)-U-box E3 Ub-ligases and the Ub-conjugating (UBC) domains of E2s showed that, among 40 rice E2s, 11 E2s accounted for 70% of the interactions with 17 ARM-U-box E3s. Thus, a single E2 could interact with multiple ARM-U-box E3s, suggesting the presence of E2 hubs for E2–E3 interactions in rice. Rice SPL11 ARM-U-box E3 displayed distinct self-ubiquitination patterns, including poly-ubiquitination, mono-ubiquitination, or no ubiquitination, depending on different E2 partners. This suggests that the mode of ubiquitination of SPL11 E3 is critically influenced by individual E2s.

Identification of a Non-covalent Ternary Complex Formed by PIAS1, SUMO1, and UBC9 Proteins Involved in Transcriptional Regulation

Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems.

Effects of 17ß-Estradiol on the Acute Necrotizing Pancreatitis after Onset in Rats

The aim of this study was to investigate the influence of 17ß-estradiol (E2) on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. Rats were divided into six groups as sham + saline, sham + single dose E2 (SDE2), sham + multiple dose E2 (MDE2), ANP + saline, ANP + SDE2, and ANP + MDE2. ANP in rats was induced by glycodeoxycholic acid. The extent of acinar cell injury, mortality, systemic cardiorespiratory variables, functional capillary density (FCD), renal/hepatic functions, and changes in some enzyme markers for pancreatic and lung tissue were investigated during ANP in rats. The induction of ANP resulted in a significant increase in the mortality rate, pancreatic necrosis, and serum activity of amylase, alanine aminotransferase (ALT), interleukin (IL)-6, lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, serum concentration of urea, and tissue activity of myeloperoxidase (MPO) and malondialdehyde (MDA) in the pancreas and lung, and a significant decrease in concentrations of calcium, blood pressure, urine output, p02, and functional capillary density (FCD). The use of E2 did not alter these changes. E2 demonstrated no effect on the course of ANP in rats. Therefore, it has no value in the treatment during acute pancreatitis.

UHRF2, a Ubiquitin E3 Ligase, Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.

Antioxidant responses of two microalgae, Selenastrum capricornutum and Chlorella sp., to estradiol and ethinylestradiol

The cellular characteristics and antioxidant responses including reactive oxygen species (ROS), esterase activity, superoxide dismutase, peroxidase (POD), glutathione (GSH), and malondialdehyde (MDA) content of two microalgal species, Selenastrum capricornutum and Chlorella sp., exposed to single and mixed estradiol (E2) and ethinlyestradiol (EE2) at a final concentration of 200 μg L−1 were investigated. The growth, autofluorescence, cell complexity, and cell size of S. capricornutum and Chlorella sp. were not affected by single and mixed estrogen treatments, but the temporal trends of these two species were different. The temporal changes of the esterase activity and ROS level in these two species were also very different, with a continuous increase with time in Chlorella but peak on Day 2 in S. capricornutum. The esterase activity of S. capricornutum was not affected by estrogen treatments; however, the activity in treated Chlorella decreased significantly from Day 2 onwards. The single EE2 and mixed E2 and EE2 treatments induced more ROS of S. capricornutum than the single E2 treatment and control on Day 4, but no other difference was found between treatments and control of Chlorella on that day. The other oxidative damage responses of S. capricornutum were not affected by estrogen treatments, except MDA increased in EE2 treatment, on the other hand, POD and GSH of Chlorella increased in both single and mixed estrogen treatments on Day 4. These results suggested that microalgae species were tolerant to E2 and EE2, but some species-specific changes occurred to combat the oxidative stress posed by estrogens.

Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry.

The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

Temporal window in which exposure to estradiol permanently modifies ovarian function causing polycystic ovary morphology in rats

To investigate the developmental window in which E(2) exposure produces irreversible changes in ovarian function resulting in polycystic ovary. Basic experimental study. University animal laboratory. Thirty Sprague-Dawley rats were administered a single E(2) valerate dose (10 mg/kg of weight) at 1, 7, 14, 21, or 30 days of age. Control rats were injected with the vehicle at 1 day of age. All rats were sacrificed at 6 months of age. Observation of vaginal opening, estrous cyclicity by vaginal smears, and ovarian morphometry in the 6-month-old rat. Measurement of ovarian noradrenaline by high-performance liquid chromatography coupled with electrochemical detection, serum levels of LH by enzyme-linked immunoassay, P, androstenedione, and E(2) by enzyme immunoassay. Rats exposed to E(2) at 1, 7, or 14 days of life did not show estrual cycling activity and maintained a polycystic ovary (PCO) condition throughout the entirety of the study. However, if the exposure to E(2) occurred after postnatal day 21, the PCO-induced condition was reversible. In rats that developed a permanent PCO condition, we observed significant effects of E(2) on ovarian morphology if exposure occurred on postnatal day 1 and a presumable effect on the hypothalamus if the exposure occurred between postnatal days 1 and 14. Our findings suggest that in rats, the most sensitive period for the promotion of an irreversible PCO morphology by estrogenic compounds is during neonatal early follicular development.

PWE-061 Homozyogosity in the ApoE4 polymorphism is associated with dysphagic symptoms in an otherwise healthy elderly population

IntroductionApolipoprotein E (ApoE) is thought to be the most common genetic risk factor for Alzheimer's disease and has a number of polymorphisms. While the E2 allele is proposed to play...

Attenuation of Chikungunya Virus Vaccine Strain 181/Clone 25 Is Determined by Two Amino Acid Substitutions in the E2 Envelope Glycoprotein

Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.

Postlesion Estradiol Treatment Increases Cortical Cholinergic Innervations via Estrogen Receptor-α Dependent Nonclassical Estrogen Signaling in Vivo

17β-Estradiol (E2) treatment exerts rapid, nonclassical actions via intracellular signal transduction system in basal forebrain cholinergic (BFC) neurons in vivo. Here we examined the effect of E2 treatment on lesioned BFC neurons in ovariectomized mice and the role of E2-induced nonclassical action in this treatment. Mice given an N-methyl-d-aspartic acid (NMDA) injection into the substantia innominata-nucleus basalis magnocellularis complex (SI-NBM) exhibited cholinergic cell loss in the SI-NBM and ipsilateral cholinergic fiber loss in the cortex. A single injection of E2 after NMDA lesion did not have an effect on cholinergic cell loss in the SI-NBM, but it restored the ipsilateral cholinergic fiber density in the cortex in a time- and dose-dependent manner. The most effective cholinergic fiber restoration was observed with 33 ng/g E2 treatment at 1 h after NMDA lesion. The E2-induced cholinergic fiber restoration was absent in neuron-specific estrogen receptor-α knockout mice in vivo. Selective activation of nonclassical estrogen signaling in vivo by estren induced E2-like restorative actions. Selective blockade of the MAPK or protein kinase A pathway in vivo prevented E2's ability to restore cholinergic fiber loss. Finally, studies in intact female mice revealed an E2-induced restorative effect that was similar to that of E2-treated ovariectomized mice. These observations demonstrate that a single E2 treatment restores the BFC fiber loss in the cortex, regardless of endogenous E2 levels. They also reveal the critical role of nonclassical estrogen signaling via estrogen receptor-α and protein kinase A-MAPK pathways in E2-induced restorative action in the cholinergic system in vivo.

E2 Conjugating Enzyme Selectivity and Requirements for Function of the E3 Ubiquitin Ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.