A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation.

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.

Factors Influencing the Sharing of Personal Health Data Based on the Integrated Theory of Privacy Calculus and Theory of Planned Behaviors Framework: Results of a Cross-Sectional Study of Chinese Patients in the Yangtze River Delta.

BACKGROUND: The health care system in China is fragmented, and the distribution of high-quality resources remains uneven and irrational. Information sharing is essential to the development of an integrated health care system and maximizing its benefits. Nevertheless, data sharing raises concerns regarding the privacy and confidentiality of personal health information, which affect the willingness of patients to share information. OBJECTIVE: This study aims to investigate patients' willingness to share personal health data at different levels of maternal and child specialized hospitals in China, to propose and test a conceptual model to identify key influencing factors, and to provide countermeasures and suggestions to improve the level of data sharing. METHODS: A research framework based on the Theory of Privacy Calculus and the Theory of Planned Behavior was developed and empirically tested through a cross-sectional field survey from September 2022 to October 2022 in the Yangtze River Delta region, China. A 33-item measurement instrument was developed. Descriptive statistics, chi-square tests, and logistic regression analyses were conducted to characterize the willingness of sharing personal health data and differences by sociodemographic factors. Structural equation modeling was used to assess the reliability and validity of the measurement as well as to test the research hypotheses. The STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) checklist for cross-sectional studies was applied for reporting results. RESULTS: The empirical framework had a good fit with the chi-square/degree of freedom (χ2/df)=2.637, root-mean-square residual=0.032, root-mean-square error of approximation=0.048, goodness-of-fit index=0.950, and normed fit index=0.955. A total of 2060 completed questionnaires were received (response rate: 2060/2400, 85.83%). Moral motive (ß=.803, P<.001), perceived benefit (ß=.123, P=.04), and perceived effectiveness of government regulation (ß=.110, P=.001) had a significantly positive association with sharing willingness, while perceived risk (ß=-.143, P<.001) had a significant negative impact, with moral motive having the greatest impact. The estimated model explained 90.5% of the variance in sharing willingness. CONCLUSIONS: This study contributes to the literature on personal health data sharing by integrating the Theory of Privacy Calculus and the Theory of Planned Behavior. Most Chinese patients are willing to share their personal health data, which is primarily motivated by moral concerns to improve public health and assist in the diagnosis and treatment of illnesses. Patients with no prior experience with personal information disclosure and those who have tertiary hospital visits were more likely to share their health data. Practical guidelines are provided to health policy makers and health care practitioners to encourage patients to share their personal health information.

Structural basis for Dicer-like function of an engineered RNase III variant and insights into the reaction trajectory of two-Mg2+-ion catalysis.

The RNase III family of dsRNA-specific endonucleases is exemplified by prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. Structures of Aquifex aeolicus RNase III (AaRNase III) and Saccharomyces cerevisiae Rnt1p (ScRnt1p) show that both enzymes recognize substrates in a sequence-specific manner and propel RNA hydrolysis by two-Mg2+-ion catalysis. Previously, we created an Escherichia coli RNase III variant (EcEEQ) by eliminating the sequence specificity via protein engineering and called it bacterial Dicer for the fact that it produces heterogeneous small interfering RNA cocktails. Here, we present a 1.8-Å crystal structure of a postcleavage complex of EcEEQ, representing a reaction state immediately after the cleavage of scissile bond. The structure not only establishes the structure-and-function relationship of EcEEQ, but also reveals the functional role of a third Mg2+ ion that is involved in RNA hydrolysis by bacterial RNase III. In contrast, the cleavage site assembly of ScRnt1p does not contain a third Mg2+ ion. Instead, it involves two more amino acid side chains conserved among eukaryotic RNase IIIs. We conclude that the EcEEQ structure (this work) represents the cleavage assembly of prokaryotic RNase IIIs and the ScRnt1p structure (PDB: 4OOG), also determined at the postcleavage state, represents the cleavage assembly of eukaryotic RNase IIIs. Together, these two structures provide insights into the reaction trajectory of two-Mg2+-ion catalysis by prokaryotic and eukaryotic RNase III enzymes.

Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection.

Ribonuclease III (RNase III) enzymes are a family of double-stranded RNA (dsRNA)-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III enzymes have been well characterized, but how eukaryotic RNase IIIs work is less clear. Here, we describe the structure of the Saccharomyces cerevisiae RNase III (Rnt1p) postcleavage complex and explain why Rnt1p binds to RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a structural motif located at the end of the Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domains (NTDs) of Rnt1p function as two rulers that measure the distance between the tetraloop and the cleavage site. These findings provide a framework for understanding eukaryotic RNase IIIs.

Effect of PM2.5 exposure on Vitamin D status among pregnant women: A distributed lag analysis.

BACKGROUND: Serum vitamin D levels are associated with exposure to air pollution, however, the lagged effect of exposure to air pollution remains unknown in pregnant women. METHODS: Pregnant women who delivered at a maternity center in Shanghai, China, from 2015 to 2019 were included in the present study. The concentration of particulate matter 2.5 (PM2.5) before 25-Hydroxyvitamin D [25(OH)D] detection was estimated using the satellite-based grid models. The distributed lag non-linear models were performed to examine the lagged association between weekly-specific PM2.5 exposure and vitamin D deficiency (VDD) or serum 25(OH)D levels. RESULTS: Among the 58,025 pregnant women included in the study (mean age at conception, 30.77 ± 3.75 years; mean prepregnancy BMI, 21.09 ± 2.55 kg/m2), 61.32% were diagnosed with VDD. Weekly-specific PM2.5 exposure at weeks 1-10 before the detection of 25(OH)D was significantly associated with an increased incidence of VDD (p < 0.05). For every 10 µg/m3 increase in PM2.5 exposure, the serum 25(OH)D level decreased by 1.346 nmol/L (95%CI: 1.183-1.508 nmol/L). The association between average PM2.5 exposure and VDD at 1-10 weeks was more significant in weather conditions with low mean sunshine hours (OR: 1.246, 95%CI: 1.221-1.271). CONCLUSION: Our study provided suggestive evidence that PM2.5 exposure at 1-10 weeks before the 25(OH)D detection may decrease the circulating 25(OH)D levels in pregnant women and increase VDD risk in pregnant women. More attention should be paid to the long-term impact of PM2.5, in particular, during weather conditions with a relatively short duration of sunshine.

Associations of PM2.5 exposure with blood glucose impairment in early pregnancy and gestational diabetes mellitus.

Exposure to fine particulate matter (PM2.5) during pregnancy has been linked to the risk of gestational diabetes mellitus (GDM), while conclusions are inconsistent. In this study we aimed to estimate the effects of prenatal PM2.5 exposure with blood glucose in early pregnancy and the GDM risk. Participants were recruited from the SH-IPMCH-BTH cohort (n = 41,929), a study of air pollution and birth outcome. All participants provided serum samples for analyses of fasting blood glucose (FBG) and HbA1c during early pregnancy. GDM was diagnosed using an oral glucose tolerance test (OGTT) with the time interval of 1 h. Prenatal exposure to PM2.5 was estimated using gap-filled satellite exposure assessments in Shanghai, China. Both FBG and HbA1c levels were significantly and positively associated with PM2.5 exposure during early pregnancy. A 10 µg/m3 increase of PM2.5 exposure from early to middle pregnancy was associated with the risk of GDM (first trimester OR=1.09, 95% CI: 1.02, 1.16; second trimester OR=1.09, 95% CI: 1.03, 1.16; first two trimester OR=1.15, 95%CI: 1.04, 1.28). The combined effects were greater among elevated FBG and HbA1c women with higher PM2.5 exposure in middle trimester (P for interaction=0.037 and 0.001, respectively). This study found that exposure to PM2.5 exposure in the 1st and 2nd trimesters was related to GDM. FBG and HbA1c played roles in the relationship between PM2.5 exposure in the 2nd trimester and GDM.

RNase III: Genetics and function; structure and mechanism.

RNase III is a global regulator of gene expression in Escherichia coli that is instrumental in the maturation of ribosomal and other structural RNAs. We examine here how RNase III itself is regulated in response to growth and other environmental changes encountered by the cell and how, by binding or processing double-stranded RNA (dsRNA) intermediates, RNase III controls the expression of genes. Recent insight into the mechanism of dsRNA binding and processing, gained from structural studies of RNase III, is reviewed. Structural studies also reveal new cleavage sites in the enzyme that can generate longer 3' overhangs.

IFITM1, CD10, SMA, and h-caldesmon as a helpful combination in differential diagnosis between endometrial stromal tumor and cellular leiomyoma.

BACKGROUND: The differential diagnosis of endometrial stromal tumor (EST) and uterine cellular leiomyoma (CL) remains a challenge in clinical practice, especially low grade endometrial stromal sarcoma (ESS) and CL, suggesting the need for novel immunomarkers panels for differential diagnosis. Interferon-induced transmembrane protein 1 (IFITM1) is a novel immunomarker for endometrial stromal cells, h-caldesmon is an immunomarker for smooth muscle cells and has a higher specificity than smooth muscle actin (SMA). So this study aimed to evaluate whether IFITM1, cluster of differentiation 10(CD10), SMA, and h-caldesmon are useful biomarker combinations for the differential diagnosis of EST and CL. METHODS: Tissue microarrays were used to detect IFITM1, CD10, SMA, and h-caldesmon immunohistochemical staining in 30 EST and 33 CL cases. RESULTS: The expressions of IFITM1 and CD10 were high in EST (86.7 and 63.3%, respectively) but low in CL (18.2 and 21.2%), whereas those of h-caldesmon and SMA were high in CL (87.9 and 100%) and low in EST (6.9 and 40%). In diagnosing EST, IFITM1 shows better sensitivity and specificity (86.7 and 81.8%, respectively) than CD10 (63.3 and 78.8%). The specificity of h-caldesmon in diagnosing CL was significantly higher (93.1%) than that of SMA (60%). When all four antibodies were combined for the differential diagnosis, the area-under-the-curve (AUC) predictive value was 0.995. The best combination for diagnosing EST was IFITM1 (+) or CD10 (+) and h-caldesmon (-) (sensitivity 86.7%, specificity 93.9%). CONCLUSION: The best combination for diagnosing CL were h-caldesmon (+) and SMA (+) (sensitivity 87.9%, specificity 100%). IFITM1, CD10, SMA, and h-caldesmon are a good combination for the differential diagnosis of EST and CL.

Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: Transition state analogs for high affinity binding.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but absent in mammals; therefore, it is an attractive target for developing novel antimicrobial agents. Previously, based on our studies of the structure and mechanism of HPPK, we created first-generation bisubstrate inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed second-generation inhibitors by replacing the phosphate bridge with a linkage that contains a piperidine moiety. Here, we report third-generation inhibitors designed based on the piperidine-containing inhibitor, mimicking the transition state. We synthesized two such inhibitors, characterized their protein-binding and enzyme inhibition properties, and determined their crystal structures in complex with HPPK, advancing the development of such bisubstrate analog inhibitors.

Influence of ambient temperature and diurnal temperature variation on the premature rupture of membranes in East China: A distributed lag nonlinear time series analysis.

BACKGROUND: Extreme ambient temperature has an adverse effect on pregnancy outcomes, but the conclusions have been inconsistent. The influence of ambient temperature and diurnal temperature variation on the premature rupture of membranes (PROM) needs further study. METHODS AND FINDINGS: The daily data of PROMs, daily meteorological and air pollutant were obtained. After controlling for potential confounding factors, the quasi-Poisson generalized additive model (GAM) combined with the distributed lag nonlinear model (DLNM) was used to analyze the association between temperature or diurnal temperature variation and PROM, including preterm premature rupture of membranes (PPROM) and term premature rupture of membranes (term PROM). Compared with the median temperature(18.7 °C), the mean temperature of 5-7 days lagging beyond 31.5 °C and below -1.5 °C was positively correlated with PROM; the mean temperature had more sensitive effect on the term PROM. Exposure to extremely high temperatures (97.5th percentile, 32 °C) had a 6-day lagging relative risk (RR) (95% CI: 1.005-1.160) of 1.08 for PROM and a 6-day lagging RR of 1.079 (95% CI: 1.005-1.159) for term PROM; Exposure to a high diurnal temperature variation (diurnal temperature variation greater than 16 °C) was positively correlated with the term PROM. Compared with the 2.5th percentile diurnal temperature variation (2 °C), exposure to the 95th percentile diurnal temperature variation (17 °C) significantly increased the risk of term PROM (RR: 1.229, 95% CI: 1.029-1.467). CONCLUSIONS: Exposure to a high-temperature and a high diurnal temperature variation environment will increase the relative risks of PROM. For pregnant women in the 3rd trimester, it is important to reduce exposure to extremely high-temperatures and greater diurnal temperature changes.

A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms.

Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the "backside" of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and RING-dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This demonstrates that an essential component of E3 complexes can simultaneously bind to E2 and enhance its loading with ubiquitin. These findings provide mechanistic insights into how ubiquitination can be stimulated.

The molecular mechanism of dsRNA processing by a bacterial Dicer.

Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

Overproduction of a Dominant Mutant of the Conserved Era GTPase Inhibits Cell Division in Escherichia coli.

Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known in Escherichia coli that the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wild-type Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphic ftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling, ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring.IMPORTANCE All cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit in Escherichia coli, but its role in regulating E. coli cell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

Nitrogen dioxide air pollution and preterm birth in Shanghai, China.

BACKGROUND: Nitrogen dioxide (NO2) is a typical indicator of traffic-related air pollution, and few studies with exposure assessment of high resolution have been conducted to explore its association with preterm birth in China. OBJECTIVES: To investigate the association between NO2 exposure based on a land use regression (LUR) model and preterm birth in Shanghai, China. METHODS: A retrospective cohort study was performed among 25,493 singleton pregnancies in a major maternity hospital in Shanghai, China, from 2014 to 2015. A temporally adjusted LUR model was used to predict the prenatal exposure to NO2 based on residence address of each gravida. Logistic regression was performed to evaluate the associations of ambient NO2 exposure with preterm birth during six exposure periods, including the entire pregnancy, the first trimester, the second trimester, the third trimester, the last month, and the last week before delivery. Sensitivity analysis with a matched case-control design was conducted to test the robustness of the association between NO2 exposure and preterm birth. RESULTS: The average NO2 concentrations during the entire pregnancy was 48.23 µg/m3 among all participants. A 10 µg/m3 increase in NO2 concentrations was associated with preterm birth, with an adjusted odds ratio of 1.03 (95% confidence interval [CI]: 0.96,1.10) for exposures during the entire pregnancy, 1.00 (95%CI: 0.95,1.06) in the first trimester, 1.01 (95%CI: 0.96,1.07) in the second trimester, 1.07 (95%CI: 1.02,1.13) in the third trimester, 1.10 (95%CI: 1.04,1.15) and 1.05 (95%CI: 1.00,1.09) in the month and week before delivery, respectively. The results of the matched case-control analysis were generally consistent with those of main analyses. CONCLUSION: NO2 may increase the risk of preterm birth, especially for exposures during the third trimester, the month and the week before delivery in Shanghai, China.

Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine.

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING-E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination.

Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78.

The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase: BIOCHEMICAL AND STRUCTURAL STUDIES.

Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN in the absence or presence of RNAP and the solution structures of RapA and RapAΔN either ligand-free or in complex with RNAP. Determined by small-angle x-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ(70) on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B.

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.

Structural insights into cell cycle control by essential GTPase Era.

Era (Escherichia coli Ras-like protein), essential for bacterial cell viability, is composed of an N-terminal GTPase domain and a C-terminal KH domain. In bacteria, it is required for the processing of 16S ribosomal RNA (rRNA) and maturation of 30S (small) ribosomal subunit. Era recognizes 10 nucleotides (1530GAUCACCUCC1539) near the 3' end of 16S rRNA and interacts with helix 45 (h45, nucleotides 1506-1529). GTP binding enables Era to bind RNA, RNA binding stimulates Era's GTP-hydrolyzing activity, and GTP hydrolysis releases Era from matured 30S ribosomal subunit. As such, Era controls cell growth rate via regulating the maturation of the 30S ribosomal subunit. Ribosomes manufacture proteins in all living organisms. The GAUCA sequence and h45 are highly conserved in all three kingdoms of life. Homologues of Era are present in eukaryotic cells. Hence, the mechanism of bacterial Era action also sheds light on the cell cycle control of eukaryotes.

Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ß-tubulin.

Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of ß-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of ß-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of ß-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.