Chemical Protein Polyubiquitination Reveals the Role of a Noncanonical Polyubiquitin Chain in DNA Damage Tolerance

Polyubiquitination of proteins regulates a variety of cellular processes, including protein degradation, NF-κB pathway activation, apoptosis, and DNA damage tolerance. Methods for generating polyubiquitinated protein with defined ubiquitin chain linkage and length are needed for an in-depth molecular understanding of protein polyubiquitination. However, enzymatic protein polyubiquitination usually generates polyubiquitinated proteins with mixed chain lengths in a low yield. We report herein a new chemical approach for protein polyubiquitination with a defined ubiquitin chain length and linkage under a mild condition that preserves the native fold of the target protein. In DNA damage tolerance, K63-polyubiquitinated proliferating cell nuclear antigen (PCNA) plays an important yet unclear role in regulating the selection of the error-free over error-prone lesion bypass pathways. Using the chemically polyubiquitinated PCNA, we revealed a mechanism of the K63 polyubiquitin chain on PCNA in promoting the error-free lesion bypass by suppressing the DNA translesion synthesis (TLS)

Transient Kinetic Analysis of USP2-Catalyzed Deubiquitination Reveals a Conformational Rearrangement in the K48-Linked Diubiquitin Substrate

Deubiquitination has emerged as an essential regulatory mechanism of a number of cellular processes. An in-depth understanding of deubiquitinating enzyme (DUB) catalysis, particularly the mode of ubiquitin binding and the individual steps in the DUB catalytic turnover, is imperative for exploiting DUBs for therapeutic intervention. In this work, we present a transient kinetic study of USP2 in hydrolyzing a model substrate Ub-AMC and a physiological substrate K48-linked diubiquitin. We conducted stopped-flow fluorescence analyses of the binding of mono- and diubiquitin to an inactive USP2 mutant and unveiled interesting differences in the binding kinetics between the two substrates. While a simple one-step binding of monoubiquitin to USP2 was observed, a biphasic binding was evident for diubiquitin. We further followed the deubiquitination reaction of Ub-AMC and K48-linked IQF-diubiquitin by USP2 using stopped-flow florescence under a single-turnover condition. Global fitting of the reaction traces revealed differences in the microscopic rate constants between Ub-AMC and the physiological diubiquitin substrate. Our binding and single-turnover data support a conformational rearrangement of the diubiquitin substrate in USP2-catalyzed deubiquitination. This finding is significant given the recent finding that the K48-linked diubiquitin is dynamic in its conformation. Our results provide useful insights into the mechanism of how USP recognizes ubiquitin moieties in a chain structure, which is important for understanding USP catalysis and developing inhibitors against USPs

Field Calibration of XAD-Based Passive Air Sampler on the Tibetan Plateau: Wind Influence and Configuration Improvement

The passive air sampler based on XAD-2 resin (XAD-PAS) has proven useful for collecting atmospheric persistent organic pollutants (POPs) in remote regions. Whereas laboratory studies have shown that, due to the open bottom of its housing, the passive sampling rate (PSR) of the XAD-PAS is susceptible to wind and other processes causing air turbulence, the sampler has not been calibrated in the field at sites experiencing high winds. In this study, the PSRs of the XAD-PAS were calibrated at three sites on the Tibetan Plateau, covering a wide range in temperature (<i>T</i>), pressure (<i>P</i>) and wind speed (<i>v</i>). At sites with low wind speeds (i.e., in a forest and an urban site), the PSRs are proportional to the ratio <i>T</i>^1.75/ <i>P</i>; at windy sites with an average wind speed above 3 m/s, the influence of <i>v</i> on PSRs cannot be ignored. Moreover, the open bottom of the XAD-PAS housing causes the PSRs to be influenced by wind angle and air turbulence caused by sloped terrain. Field calibration, wind speed measurements, and computational fluid dynamics (CFD) simulations indicate that a modified design incorporating an air spoiler consisting of 4 metal sheets dampens the turbulence caused by wind angle and sloped terrain and caps the PSR at ∼5 m³/day, irrespective of ambient wind. Therefore, the original XAD-PAS with an open bottom is suitable for deployment in urban areas and other less windy places, the modified design is preferable in mountain regions and other places where air circulation is complicated and strong

DFT/TDDFT investigation on the photophysical properties of a series of phosphorescent cyclometalated complexes based on the benchmark complex FIrpic

<p>The photophysical properties of four Ir(III) complexes have been investigated by means of the density functional theory/time-dependent density functional theory (DFT/TDDFT). The effect of the electron-withdrawing and electron-donating substituents on charge injection, transport, absorption and phosphorescent properties has been studied. The theoretical calculation shows that the lowest-lying singlet absorptions for complexes <b>1</b>–<b>4</b> are located at 387, 385, 418 and 386 nm, respectively. For <b>1</b>–<b>4</b>, the phosphorescence at 465, 485, 494 and 478 nm is mainly attributed to the LUMO → HOMO and LUMO → HOMO-1 transition configurations characteristics. In addition, ionisation potential (IP), electron affinities (EAs) and reorganisation energy have been investigated to evaluate the charge transfer and balance properties between hole and electron. The balance of the reorganisation energies for complex <b>3</b> is better than others. The difference between hole transport and electron transport for complex <b>3</b> is the smallest among these complexes, which is beneficial to achieve the hole and electron transfer balance in emitting layer.</p

Vital organ functions in both groups during XBJ treatment.

<p>Note: XBJ, <i>Xuebijing</i> injection; ALT, alanine aminotransferase; TnT, troponin T.</p><p>*<i>P</i><0.05 XBJ group <i>vs</i>. control group;</p><p>**<i>P</i><0.05 control group on Day 3 <i>vs</i>. control group on Day 1.</p><p>Vital organ functions in both groups during XBJ treatment.</p

Kaplan-Meier Survival Curves for (A) all patients and (B) patients ingesting 10–30 ml of 20% liquid paraquat (w/v).

<p>The differences in survival curves between the control and XBJ groups were not significant in A (<i>P</i> = 0.06), but significant in B (<i>P</i> = 0.02), as compared using a Log rank test. XBJ, <i>Xuebijing</i> injection.</p

Fingerprint chromatograph of XBJ.

<p>Fingerprint chromatograph of XBJ.</p

Significant risk factors for mortality among patients with paraquat poisoning.

<p>Note: Adjusted for covariates including age, gender, body mass index, time to gastric lavage, APACHE II scores, volume of oral paraquat, and time to haemoperfusion. APACHE II, Acute Physiology and Chronic Health Evaluation II; BMI, body mass index; CI, confidence interval.</p><p>Significant risk factors for mortality among patients with paraquat poisoning.</p

Gene Expression Analysis of CL-20-Induced Reversible Neurotoxicity Reveals GABA_A Receptors as Potential Targets in the Earthworm <i>Eisenia fetida</i>

The earthworm <i>Eisenia fetida</i> is one of the most used species in standardized soil ecotoxicity tests. End points such as survival, growth, and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we apply a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an <i>E. fetida</i>-specific shotgun microarray targeting 15119 unique <i>E. fetida</i> transcripts. Using this array we profiled gene expression in <i>E. fetida</i> in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm² of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at days 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control, and 13-day exposed (i.e., 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shotgun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by noncompetitively blocking the ligand-gated GABA_A receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress

Gene Expression Analysis of CL-20-Induced Reversible Neurotoxicity Reveals GABA_A Receptors as Potential Targets in the Earthworm <i>Eisenia fetida</i>

The earthworm <i>Eisenia fetida</i> is one of the most used species in standardized soil ecotoxicity tests. End points such as survival, growth, and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we apply a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an <i>E. fetida</i>-specific shotgun microarray targeting 15119 unique <i>E. fetida</i> transcripts. Using this array we profiled gene expression in <i>E. fetida</i> in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm² of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at days 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control, and 13-day exposed (i.e., 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shotgun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by noncompetitively blocking the ligand-gated GABA_A receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress