E2 Domain Research Articles

Rabex-5 E3 and Rab5 GEF domains differ in their regulation of Ras, Notch, and PI3K signaling in Drosophila wing development.

Rabex-5 (also called RabGEF1), a protein originally characterized for its Rab5 GEF function, also has an A20-like E3 ubiquitin ligase domain. We and others reported that Rabex-5 E3 activity promotes Ras mono- and di-ubiquitination to inhibit Ras signaling in Drosophila and mammals. Subsequently, we reported that Rabex-5 inhibits Notch signaling in the Drosophila hematopoietic system. Here we report genetic interactions using Rabex-5 transgenes encoding domain-specific mutations that show that Rabex-5 requires an intact E3 domain to inhibit Notch signaling in the epithelial tissue of the developing wing. Surprisingly, we discovered that Rabex-5 with an impaired E3 domain but active Rab5 GEF domain suppresses Notch loss-of-function phenotypes and enhances both Notch duplication phenotypes and activated Ras phenotypes consistent with a model that the Rab5 GEF activity of Rabex-5 might positively regulate Ras and Notch. Positive and negative regulation of developmental signaling by its different catalytic domains could allow Rabex-5 to precisely coordinate developmental signaling to fine-tune patterning. Finally, we report that Rabex-5 also inhibits the overgrowth due to loss of PTEN or activation of PI3K but not activation of AKT. Inhibition of Ras, Notch, and PI3K signaling may explain why Rabex-5 is deleted in some cancers. Paradoxically, Rabex-5 is reported to be an oncogene in other cancers. We propose that Rabex-5 acts as a tumor suppressor via its E3 activity to inhibit Ras, Notch, and PI3K signaling and as an oncogene via its Rab5 GEF activity to enhance Ras and Notch signaling.

Zinc and copper effect mechanical cell adhesion properties of the amyloid precursor protein.

The amyloid precursor protein (APP) can be modulated by the binding of copper and zinc ions. Both ions bind with low nanomolar affinities to both subdomains (E1and E2) in the extracellular domain of APP. However, the impact of ion binding on structural and mechanical trans-dimerization properties is yet unclear. Using a bead aggregation assay (BAA), we found that zinc ions increase the dimerization of both subdomains, while copper promotes only dimerization of the E1 domain. In line with this, scanning force spectroscopy (SFS) analysis revealed an increase in APP adhesion force up to three-fold for copper and zinc. Interestingly, however, copper did not alter the separation length of APP dimers, whereas high zinc concentrations caused alterations in the structural features and a decrease of separation length. Together, our data provide clear differences in copper and zinc mediated APP trans-dimerization and indicate that zinc binding might favor a less flexible APP structure. This fact is of significant interest since changes in zinc and copper ion homeostasis are observed in Alzheimer's disease (AD) and were reported to affect synaptic plasticity. Thus, modulation of APP trans-dimerization by copper and zinc could contribute to early synaptic instability in AD.

A pore locus in the E1 domain differentially regulates Cx26 and Cx30 hemichannel function

A pore locus in the E1 domain differentially regulates Cx26 and Cx30 hemichannel function

Changes in the chikungunya virus E1 glycoprotein domain II and hinge influence E2 conformation, infectivity, and virus-receptor interactions.

In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein β-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo. The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.IMPORTANCEArboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects.

Structural basis of the monkeypox virus mRNA cap N7 methyltransferase complex

ABSTRACT The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after COVID-19. The mRNA cap N7 methyltransferase (RNMT) of MPXV methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs and plays a vital role in evading host antiviral immunity. MPXV RNMT is composed of the large subunit E1 and the small subunit E12. How E1 and E12 of MPXV assembly remains unclear. Here, we report the crystal structures of E12, the MTase domain of E1 with E12 (E1CTD-E12) complex, and the E1CTD-E12-SAM ternary complex, revealing the detailed conformations of critical residues and the structural changes upon E12 binding to E1. Functional studies suggest that E1CTD N-terminal extension (Asp545-Arg562) and the small subunit E12 play an essential role in the binding process of SAM. Structural comparison of the AlphaFold2-predicted E1, E1CTD-E12 complex, and the homologous D1-D12 complex of vaccinia virus (VACV) indicates an allosteric activating effect of E1 in MPXV. Our findings provide the structural basis for the MTase activity stimulation of the E1-E12 complex and suggest a potential interface for screening the anti-poxvirus inhibitors.

Dimerization of a 5-kDa domain defines the architecture of the 5-MDa gammaproteobacterial pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.

Molecular dynamics simulation of apolipoprotein E3 lipid nanodiscs

Nanodiscs are binary discoidal complexes of a phospholipid bilayer circumscribed by belt-like helical scaffold proteins. Using coarse-grained and all-atom molecular dynamics simulations, we explore the stability, size, and structure of nanodiscs formed between the N-terminal domain of apolipoprotein E3 (apoE3-NT) and variable number of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) molecules. We study both parallel and antiparallel double-belt configurations, consisting of four proteins per nanodisc. Our simulations predict nanodiscs containing between 240 and 420 DMPC molecules to be stable. The antiparallel configurations exhibit an average of 1.6 times more amino acid interactions between protein chains and 2 times more ionic contacts, compared to the parallel configuration. With one exception, DMPC order parameters are consistently larger in the antiparallel configuration than in the parallel one. In most cases, the root mean square deviation of the positions of the protein backbone atoms is smaller in the antiparallel configuration. We further report nanodisc size, thickness, radius of gyration, and solvent accessible surface area. Combining all investigated parameters, we hypothesize the antiparallel protein configuration leading to more stable and more rigid nanodiscs than the parallel one.

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

A small molecule targeting the interaction between human papillomavirus E7 oncoprotein and cellular phosphatase PTPN14 exerts antitumoral activity in cervical cancer cells

Human papillomavirus (HPV)-induced cancers still represent a major health issue for worldwide population and lack specific therapeutic regimens. Despite substantial advancements in anti-HPV vaccination, the incidence of HPV-related cancers remains high, thus there is an urgent need for specific anti-HPV drugs. The HPV E7 oncoprotein is a major driver of carcinogenesis that acts by inducing the degradation of several host factors. A target is represented by the cellular phosphatase PTPN14 and its E7-mediated degradation was shown to be crucial in HPV oncogenesis. Here, by exploiting the crystal structure of E7 bound to PTPN14, we performed an in silico screening of small-molecule compounds targeting the C-terminal CR3 domain of E7 involved in the interaction with PTPN14. We discovered a compound able to inhibit the E7/PTPN14 interaction in vitro and to rescue PTPN14 levels in cells, leading to a reduction in viability, proliferation, migration, and cancer-stem cell potential of HPV-positive cervical cancer cells. Mechanistically, as a consequence of PTPN14 rescue, treatment of cancer cells with this compound altered the Yes-associated protein (YAP) nuclear-cytoplasmic shuttling and downstream signaling. Notably, this compound was active against cervical cancer cells transformed by different high-risk (HR)-HPV genotypes indicating a potential broad-spectrum activity. Overall, our study reports the first-in-class inhibitor of E7/PTPN14 interaction and provides the proof-of-principle that pharmacological inhibition of this interaction by small-molecule compounds could be a feasible therapeutic strategy for the development of novel antitumoral drugs specific for HPV-associated cancers.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of β-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

Q531L mutation in the capsid protein of hepatitis E virus genotype 1 causes infections in patients with altered immunity and immunosuppressive condition: Mechanism based on wet lab and in-silico findings

ObjectivesThe study aimed to observe the effects of Q531L mutation in the E2s domain of HEV ORF2 on the circulation of the virus among the immunocompetent patients, diabetic patients (immunosuppressive), and pregnant women (altered immunity). MethodsThirty two HEV-RNA samples from pregnant and non-pregnant females were subjected to real time (RT)-qPCR, conventional RT-PCR and Sanger sequencing of HEV ORF2 region covering immunodominant E2s domain (459–606 aa). Liver functions tests using serum samples of the patients were performed. In-silico prediction of CD8+ cytotoxic T lymphocytes (CTL) epitope covering the 531st residue of HEV genotype 1 ORF2/E2s was performed. ResultsThree out of 32 samples harbored a nonsynonymous substitution, resulting in HEV genotype 1 ORF2 531Gln>Leu. The patients with HEV 531Gln had significantly higher serum ALT levels than 531Leu (P = 0.001) and in addition, the former had 173-fold higher viral load than the latter (1,360,869.45 ± 5,298,899.45 vs 784 ± 507.73). The HLA-mutant peptide complexes had higher binding affinities than the HLA-wild type complexes, showing Cluspro2.0/PRODIGY docking scores(Kcal/mol)) of −724.6/−9.2 and −854.2/−9.8 versus −680.1/−8.2 and −757.7/−7.5, respectively. One of the three 531Leu patients was a non-pregnant woman with diabetes and the rest 2 were pregnant. ConclusionsThe hosts with immunosuppressive/altered immunity may provide conducive niches for HEV 531Leu.

Characterization of the role of Kunitz-type protease inhibitor domain in dimerization of amyloid precursor protein.

A major difference between amyloid precursor protein (APP) isoforms (APP695 and APP751) is the existence of a Kunitz type protease inhibitor (KPI) domain which has a significant impact on the homo- and hetero-dimerization of APP isoforms. However, the exact molecular mechanisms of dimer formation remain elusive. To characterize the role of the KPI domain in APP dimerization, we performed a single molecule pull down (SiMPull) assay where homo-dimerization between tethered APP molecules and soluble APP molecules was highly preferred regardless of the type of APP isoforms, while hetero-dimerization between tethered APP751 molecules and soluble APP695 molecules was limited. We further investigated the domain level APP-APP interactions using coarse-grained models with the Martini force field. Though the model initial ternary complexes (KPI-E1, KPI-KPI, KPI-E2, E1-E1, E2-E2, and E1-E2) generated using HADDOCK (HD) and AlphaFold2 (AF2), the binding free energy profiles and the binding affinities of the domain combinations were investigated via the umbrella sampling with Martini force field. Additionally, membrane-bound microenvironments at the domain level were modeled. As a result, it was revealed that the KPI domain has a stronger attractive interaction with itself than the E1 and E2 domains, as reported elsewhere. Thus, the KPI domain of APP751 may form additional attractive interactions with E1, E2 and the KPI domain itself, whereas it is absent in APP695. In conclusion, we found that the APP751 homo-dimer formation is predominant than the homodimerization in APP695, which is facilitated by the presence of the KPI domain.

The second extracellular domain of connexin 50 is important for in cell adhesion, lens differentiation, and adhesion molecule expression

Connexin (Cx)-forming channels play essential roles in maintaining lens homeostasis and transparency. We showed here channel-independent roles of Cx50 in cell-cell adhesion and confirmed the second extracellular (E2) domain as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50 (E2) domain. This function is Cx channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. In addition, we generated eight site mutations of unique residues between Cx50 and the other two lens Cxs and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin and β-catenin. Expression of the adhesion-impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both invitro and invivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.

Tsunami Numerical Modelling in Ujung Kulon National Park with the Earthquake Magnitude Scenario of 6.5 and 6.9 Mw

Tsunami modelling was conducted to simulate tsunami events based on earthquake data previously occurring around Ujung Kulon National Park (TNUK), Banten Province using TUNAMI-F1 and TUNAMI-N3. TUNAMI-F1 is a modelling method to get the wave height and tsunami arrival time with a grid spacing of 0.00167°. While the TUNAMI-N3 is a model that applies the theory of wave propagation linearly in a grid variation to get a high output of tsunami inundation. The tsunami source data was used from historical earthquake generation on January 14, 2022 (6.5 Mw) and August 2, 2019 (6,9 Mw). The reverse fault type scenario from Wells & Coppersmith (1994) is used to calculate the scaling law with length, width, and the focal mechanism value obtained from the United States Geological Survey (USGS) to generate initial waves based on the multideform model. The TUNAMI-N3 model applies a nested model with variations in grid spacing and the most detailed value is 68,556 m. The tsunami wave height based on the magnitude 6.5 Mw scenario is around 0.001 m to 0.05 m with the highest wave height found on the southern coast of TNUK and the southern coastal area of Peucang Island (0.03-0.05m). The inundation produced is only visible in the E1 domain reaching 96-192 m in areas A, B, C. Then, the maximum inundation area is the south coast of TNUK (0.01-0.03m). Based on the second scenario, the height of the tsunami waves generated is in the range of 0.001 − 0.3 m. The maximum tsunami height was in the Rancecet Beach area, south of Tinjil Island, the south, east, and north coasts of TNUK (0.03-0.3m). The run-up distance resulting from scenario 1 is less than 1 km. Based on TUNAMI-F1 model results, wave height maximum caused by 6.5 Mw magnitude is higher than 6.9 Mw.

Development and application of an indirect ELISA for detecting equine IgG antibodies against Getah virus with recombinant E2 domain protein

Getah virus (GETV) disease is a mosquito-borne infectious disease that causes fever, aseptic meningitis, and abortion in a variety of animals. Currently, the epidemic trend of GETV disease increases seriously worldwide, especially in China, posing a potential threat to animal safety and public health. However, there are few reports about the epidemiological investigation of GETV disease in China as well as a lack of commercial diagnostic kit for GETV antibody. Therefore, the establishment of a rapid, sensitive and suitable GETV antibody detection method for large-scale samples is an urgent request to fully understand the prevalence of GETV disease. Here, a recombinant plasmid pET22b-GETV-E2d that contained the domain of GETV-E2 (E2d) fused to His-tag was constructed to express recombinant protein E2d (rE2d) in Escherichia coli. The rE2d was mainly expressed in inclusion bodies. And it was purified successfully by nickel affinity column so that it could be used to develop an indirect ELISA (rE2d-ELISA). After optimizing reaction conditions of rE2d-ELISA, the cut-off value was determined as 0.396 with 100 equine sera tested by virus neutralization test (VNT). Furthermore, rE2d-ELISA method showed the positive rate of IgG antibodies against GETV was 54.3% based on testing 646 clinical serum samples obtained in Xinjiang whereas the overall coincidence rate between rE2d-ELISA and VNT was 94.0%, with 98.2% sensitivity and 92.6% specificity. The findings suggest that the developed IgG ELISA employing recombinant E2d promises was an efficient and low-cost type of antibody detection method for horse, which will benefit for prevention of GETV outbreaks in horses.

Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the β-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

Genome-wide identification of RUB activating enzyme and conjugating enzyme gene families and their expression analysis under abiotic stresses in Capsicum annuum.

NEDD8/RUB, as a ubiquitin-like protein, participates in the post-translational modification of protein and requires unique E1, E2, and E3 enzymes to bind to its substrate. The RUB E1 activating enzyme and E2 conjugating enzyme play a significant role in the neddylation. However, it is unknown whether RUB E1 and E2 exist in pepper and what its function is. In this study, a total of three putative RUB E1 and five RUB E2 genes have been identified in the pepper genome. Subsequently, their physical and chemical properties, gene structure, conserved domains and motifs, phylogenetic relationship, and cis-acting elements were analyzed. The structure and conserved domain of RUB E1 and E2 are similar to that of Arabidopsis and tomato. The RUB E1 and E2 genes were randomly distributed on seven chromosomes, and there were two pairs of collinearity between pepper and Arabidopsis and eight pairs of collinearity between pepper and tomato. Phylogenetic analysis reveals that RUB E1 and E2 genes of pepper have a closer relationship with that of tomato, potato, and Nicotiana attenuate. The cis-elements of RUB E1 and E2 genes contained hormone response and stress response. RUB E1 and E2 genes were expressed in at least one tissue and CaRCE1.3 and CaRCE2.1 were exclusively expressed in flowers and anthers. Moreover, the expression of RUB E1 genes (CaECR1, CaAXR1.1, and CaAXR1.2) and RUB E2 genes (CaRCE1.1, CaRCE1.2, and CaRCE2.1) was increased to varying degrees under low-temperature, drought, salt, ABA, and IAA treatments, while CaRCE1.3 and CaRCE2.2 were down-regulated under low-temperature treatment. In addition, these genes were hardly expressed under MeJA treatment. In summary, this study provides a theoretical foundation to explore the role of RUB E1 and E2 in the response of plants to stress.

Soluble CCR2 gene therapy controls joint inflammation, cartilage damage, and the progression of osteoarthritis by targeting MCP-1 in a monosodium iodoacetate (MIA)-induced OA rat model

BackgroundOsteoarthritis (OA) is the most common type of degenerative arthritis and affects the entire joint, causing pain, joint inflammation, and cartilage damage. Various risk factors are implicated in causing OA, and in recent years, a lot of research and interest have been directed toward chronic low-grade inflammation in OA. Monocyte chemoattractant protein-1 (MCP-1; also called CCL2) acts through C–C chemokine receptor type 2 (CCR2) in monocytes and is a chemotactic factor of monocytes that plays an important role in the initiation of inflammation. The targeting of CCL2–CCR2 is being studied as part of various topics including the treatment of OA.MethodsIn this study, we evaluated the potential therapeutic effects the sCCR2 E3 gene may exert on OA. The effects of sCCR2 E3 were investigated in animal experiments consisting of intra-articular injection of sCCR2 E3 in a monosodium iodoacetate (MIA)-induced OA rat model. The effects after intra-articular injection of sCCR2 E3 (fusion protein encoding 20 amino acids of the E3 domain of the CCL2 receptor) in a monosodium iodoacetate-induced OA rat model were compared to those in rats treated with empty vector (mock treatment) and full-length sCCR2.ResultsPain improved with expression of the sCCR2 gene. Improved bone resorption upon sCCR2 E3 gene activation was confirmed via bone analyses using micro-computed tomography. Histologic analyses showed that the sCCR2 E3 gene exerted protective effects against cartilage damage and anti-inflammatory effects on joints and the intestine.ConclusionsThese results show that sCCR2 E3 therapy is effective in reducing pain severity, inhibiting cartilage destruction, and suppressing intestinal damage and inflammation. Thus, sCCR2 E3 may be a potential therapy for OA.

Generation of Allogeneic CAR T Cells through Specific Degradation of the T Cell Antigen Receptor by E3 Ubiquitin Ligase Fusion Proteins

Receptor downregulation is instrumental for many therapeutic interventions. Receptor knockout through gene-editing technologies is efficient but can introduce off-target mutations and chromothripsis. Regulation of gene expression at the protein level is a promising alternative. Here, we present results showing the targeted T cell antigen receptor (TCR) degradation using chimeric E3 fusion proteins that we call Receptor Targeting Chimeras (ReceptorTAC). We show that TCR degradation is dependent on enzymatically active, membrane-anchored E3 ligase variants. TCR specificity was achieved by direct fusion of an E3 domain to the CD3ζ transmembrane sequence. Jurkat and primary T cells stably expressing the ReceptorTAC constructs showed significantly reduced responses to TCR stimulation. We also used our ReceptorTAC technology to generate TCR-deficient, claudin18.2-specific CAR T cells, where the activity of the CAR was unaffected by the expression of the ReceptorTAC. These data indicate that our ReceptorTAC molecule can be used to generate allogeneic CAR T cells.

Functions of Papillomavirus E8^E2 Proteins in Tissue Culture and In Vivo.

Papillomaviruses (PV) replicate in undifferentiated keratinocytes at low levels and to high levels in differentiated cells. The restricted replication in undifferentiated cells is mainly due to the expression of the conserved viral E8^E2 repressor protein, a fusion protein consisting of E8 and the hinge, DNA-binding, and dimerization domain of E2. E8^E2 binds to viral genomes and represses viral transcription and genome replication by recruiting cellular NCoR/SMRT-HDAC3 corepressor complexes. Tissue culture experiments have revealed that E8^E2 modulates long-term maintenance of extrachromosomal genomes, productive replication, and immortalization properties in a virus type-dependent manner. Furthermore, in vivo experiments have indicated that Mus musculus PV1 E8^E2 is required for tumor formation in immune-deficient mice. In summary, E8^E2 is a crucial inhibitor whose levels might determine the outcome of PV infections.