Conserved Residues at the Mtr4 C-Terminus Coordinate Helicase Activity and Exosome Interactions.

Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions.

Depression, Anxiety, and Stress among Hangover-Sensitive and Hangover-Resistant Drinkers.

This study investigated potential differences in baseline (i.e., non-hangover-related) levels of depression, anxiety, and stress between individuals who are sensitive to and those resistant to hangovers after consuming alcohol. Participants included 5111 university students from the Netherlands and the U.K., including 3205 hangover-sensitive and 1906 hangover-resistant drinkers. All participants completed surveys on their demographics, alcohol consumption, and hangover susceptibility (whether they experienced a hangover in the past 12 months), as well as their baseline levels of depression, anxiety, and stress on the DASS-21 scale. The results showed that hangover-sensitive drinkers had significantly higher levels of anxiety and stress, but not depression, compared to hangover-resistant drinkers. However, the observed differences between the two groups were small, with a magnitude of less than 1 out of 42 points on the DASS-21 anxiety and stress subscales, and are thus unlikely to be clinically meaningful.

Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

Unified classification and risk-stratification in Acute Myeloid Leukemia.

Clinical recommendations for Acute Myeloid Leukemia (AML) classification and risk-stratification remain heavily reliant on cytogenetic findings at diagnosis, which are present in <50% of patients. Using comprehensive molecular profiling data from 3,653 patients we characterize and validate 16 molecular classes describing 100% of AML patients. Each class represents diverse biological AML subgroups, and is associated with distinct clinical presentation, likelihood of response to induction chemotherapy, risk of relapse and death over time. Secondary AML-2, emerges as the second largest class (24%), associates with high-risk disease, poor prognosis irrespective of flow Minimal Residual Disease (MRD) negativity, and derives significant benefit from transplantation. Guided by class membership we derive a 3-tier risk-stratification score that re-stratifies 26% of patients as compared to standard of care. This results in a unified framework for disease classification and risk-stratification in AML that relies on information from cytogenetics and 32 genes. Last, we develop an open-access patient-tailored clinical decision support tool.

Purification and characterization of Mtr4 and TRAMP from S. cerevisiae.

The Ski2-like RNA helicase, Mtr4, plays a central role in nuclear RNA surveillance pathways by delivering targeted substrates to the RNA exosome for processing or degradation. RNA target selection is accomplished by a variety of Mtr4-mediated protein complexes. In S. cerevisiae, the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex prepares substrates for exosomal decay through the combined action of polyadenylation and helicase activities. Biophysical and structural studies of Mtr4 and TRAMP require highly purified protein components. Here, we describe robust protocols for obtaining large quantities of pure, active Mtr4 and Trf4-Air2 from S. cerevisiae. The proteins are recombinantly expressed in E. coli and purified using affinity, ion exchange, hydrophobic exchange and size exclusion chromatography. Care is taken to remove nuclease contamination during the prep. Assembly of TRAMP is achieved by combining individually purified Mtr4 and Trf4-Air2. We further describe a strand displacement assay to characterize Mtr4 helicase unwinding activity.

Hydrogen-deuterium exchange mass spectrometry of Mtr4 with diverse RNAs reveals substrate-dependent dynamics and interfaces in the arch.

Mtr4 is a eukaryotic RNA helicase required for RNA decay by the nuclear exosome. Previous studies have shown how RNA en route to the exosome threads through the highly conserved helicase core of Mtr4. Mtr4 also contains an arch domain, although details of potential interactions between the arch and RNA have been elusive. To understand the interaction of Saccharomyces cerevisiae Mtr4 with various RNAs, we have characterized RNA binding in solution using hydrogen-deuterium exchange mass spectrometry, and affinity and unwinding assays. We have identified RNA interactions within the helicase core that are consistent with existing structures and do not vary between tRNA, single-stranded RNA and double-stranded RNA constructs. We have also identified novel RNA interactions with a region of the arch known as the fist or KOW. These interactions are important for RNA unwinding and vary in strength depending on RNA structure and length. They account for Mtr4 discrimination between different RNAs. These interactions further drive Mtr4 to adopt a closed conformation characterized by reduced dynamics of the arch arm and intra-domain contacts between the fist and helicase core.

An on-premise study to investigate the effects of mixing alcohol with caffeinated beverages.

OBJECTIVE: The purpose of this on-premise study was to determine if mixing alcohol with caffeinated mixers had an impact on objective and subjective intoxication. METHODS: The study was conducted across eight drinking occasions in the City of Bristol, UK. Participants (N = 1041) were recruited outside popular night-time entertainment venues and interviewed regarding their alcohol consumption for that particular evening, including whether or not they had consumed caffeinated beverages with alcohol. Subjective intoxication was rated on an 11-point scale and objective intoxication determined with a breath alcohol test. Depending on their consumption on the night of the interview, participants also reported whether they consumed alcohol mixed with caffeinated mixers or alcohol-only on other consumption occasions. RESULTS: Between-subjects analyses found that alcohol-caffeine consumers consumed more alcohol and had higher objective and subjective intoxication than those who consumed alcohol-only. These results remained significant regardless of whether or not they mixed alcohol with caffeinated mixers or consumed alcohol-only on the night of the interview. Within-subject analyses revealed that alcohol-caffeine consumers drank the same or less alcohol on alcohol-caffeine occasions compared to alcohol-only occasions. CONCLUSIONS: These findings provide support that alcohol-caffeine use does not increase overall alcohol consumption, and may be one manifestation of a high risk-taking personality.

A Comparison Between Ecological Momentary Assessment and the Adapted-Quick Drinking Screen: Alcohol Mixed With Energy Drinks.

AIMS: To compare alcohol consumption and risk-taking behaviours on alcohol mixed with energy drink (AMED) and alcohol-only (AO) drinking occasions collected via ecological momentary assessment (EMA) versus retrospective survey methods (adapted-Quick Drinking Screen: a-QDS). METHODS: Completing participants were 52 university students who reported AMED consumption during the 30-day data collection period. Alcohol consumption and risk-taking behaviours were captured for recreational AMED and AO consumption occasions using a smartphone-based app across 30 days. Data were aggregated for comparison with the a-QDS conducted at the end of data collection. RESULTS: Irrespective of data collection method, alcohol was consumed more frequently and at higher quantities on the heaviest drinking occasions when consumed alone compared with when it was mixed with energy drinks. Consistent with this finding, more risk-taking behaviours were experienced on AO occasions compared with AMED occasions. Compared with the a-QDS, the quantity of alcohol consumed on the average and heaviest drinking occasion was significantly higher when reported via EMA. This was consistent across both AO and AMED drinking occasions. CONCLUSION: EMA provides a more valid measure of consumption quantity compared with retrospective recall, which was susceptible to under-reporting, although this was not differentially affected across consumption occasions. In line with previous research, this study demonstrated that mixing alcohol with energy drinks does not increase alcohol consumption or risk-taking behaviours.

Significant Loop Motions in the SsoPTP Protein Tyrosine Phosphatase Allow for Dual General Acid Functionality.

Conformational dynamics are important factors in the function of enzymes, including protein tyrosine phosphatases (PTPs). Crystal structures of PTPs first revealed the motion of a protein loop bearing a conserved catalytic aspartic acid, and subsequent nuclear magnetic resonance and computational analyses have shown the presence of motions, involved in catalysis and allostery, within and beyond the active site. The tyrosine phosphatase from the thermophilic and acidophilic Sulfolobus solfataricus (SsoPTP) displays motions of its acid loop together with dynamics of its phosphoryl-binding P-loop and the Q-loop, the first instance of such motions in a PTP. All three loops share the same exchange rate, implying their motions are coupled. Further evidence of conformational flexibility comes from mutagenesis, kinetics, and isotope effect data showing that E40 can function as an alternate general acid to protonate the leaving group when the conserved acid, D69, is mutated to asparagine. SsoPTP is not the first PTP to exhibit an alternate general acid (after VHZ and TkPTP), but E40 does not correspond to the sequence or structural location of the alternate general acids in those precedents. A high-resolution X-ray structure with the transition state analogue vanadate clarifies the role of the active site arginine R102, which varied in structures of substrates bound to a catalytically inactive mutant. The coordinated motions of all three functional loops in SsoPTP, together with the function of an alternate general acid, suggest that catalytically competent conformations are present in solution that have not yet been observed in crystal structures.

Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases.

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a noncatalytic residue in the WPD-loops of YopH and PTP1B result in shifted pH-rate profiles from an altered kinetic pK a of the nucleophilic cysteine. Compared to wild type, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations but suggest an increased preference for the WPD-loop-closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pK a values of catalytic residues by nonchemical processes affords a means for nature to alter an enzyme's pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.

A Cross-Cultural Comparison of the Effects of Alcohol Mixed with Energy Drink (AMED) Consumption on Overall Alcohol Consumption and Related Consequences.

There is a growing body of scientific literature examining the effects of alcohol mixed with energy drink (AMED) on alcohol consumption and related negative consequences, such as risky behavior or negative health effects. It is unknown whether differences in cultural context may influence these AMED effects. The current cross-cultural study compared the data of N = 6881 students from The Netherlands (N = 4424), UK (N = 1594), and Australia (N = 863). Demographics, alcohol consumption, its consequences, and motives for AMED consumption were assessed. Analyses included (a) between-groups comparison of AMED and alcohol only (AO) consumers, (b) within-subjects comparison of AMED and AO occasions among AMED consumers only, and (c) comparisons between the three countries. The between-groups analysis revealed that AMED consumers drink more alcohol than AO consumers (p < 0.001). AMED consumers differed from AO consumers in many other aspects. For example, AMED consumers were significantly more often a smoker and had higher risk-taking scores. Within subject analysis among AMED consumers showed that significantly less alcohol was consumed on AMED, compared to AO occasions (p < 0.001). These findings were observed for both typical drinking occasions and the past month's heaviest drinking occasion, and were consistent across the three countries. Comparisons between countries revealed that on both AMED and AO occasions, the UK sample consumed significantly more alcohol than the Australian and Dutch samples. Across countries, neutral motives such as 'I like the taste' and 'I wanted to drink something else' were the most frequently reported motives for consuming AMED. The most notable difference between the countries was the finding that consuming AMED 'To get drunk' was endorsed significantly more often among the UK sample (45.6%) than the Australian (31.2%) and Dutch (8.0%) samples. Negative alcohol-related consequences were significantly less frequently reported for AMED occasions compared to AO occasions. Some country-specific consequences of AMED consumption were observed, but these were more likely related to characteristics of the country and their drinking culture (e.g., the Australian sample reported more often driving a car after AMED consumption compared to the other countries, and this pattern of results was also found for AO occasions). In conclusion, there were limited differences between countries with regard to demographics of consumers and motives for AMED consumption, but the UK sample consumed significantly more alcohol and reported the highest frequency of negative alcohol related consequences. Consistent across countries was the observation that AMED consumers consume significantly less alcohol on their past month heaviest AMED drinking occasion, compared to their past month heaviest AO drinking occasion.

Risk-Taking Behavior and the Consumption of Alcohol Mixed with Energy Drink among Australian, Dutch and UK Students.

The relationship between risk-taking behavior, alcohol consumption and negative alcohol-related consequences is well known. The current analyses were conducted to investigate whether alcohol mixed with energy drink (AMED) is related to risk-taking behavior and if there is a relationship between the amount of energy drink mixed with alcohol consumed, risk-taking behavior and negative alcohol-related consequences. Data from N = 1276 AMED consuming students from the Netherlands, UK and Australia who completed the same survey were evaluated. The analysis revealed that, compared to AMED occasions, on alcohol only (AO) occasions significantly more alcohol was consumed and significantly more negative alcohol-related consequences were reported. On both AO and AMED occasions, there was a strong and positive relationship between amount of alcohol consumed, level of risk-taking behavior and number of reported negative alcohol-related consequences. In contrast, the level of risk-taking behavior was not clearly related to energy drink consumption. Across risk-taking levels, differences in the amount of energy drink consumed on AMED occasions did not exceed one 250 mL serving of energy drink. When correcting for the amount of alcohol consumed, there were no statistically significant differences in the number of energy drinks consumed on AMED occasions between the risk-taking groups. In conclusion, alcohol consumption is clearly related to risk-taking behavior and experiencing negative alcohol-related consequences. In contrast, energy drink intake was not related to level of risk-taking behavior and only weakly related to the number of experienced negative alcohol-related consequences.

Mtr4 RNA helicase structures and interactions.

Mtr4 is a Ski2-like RNA helicase that plays a central role in RNA surveillance and degradation pathways as an activator of the RNA exosome. Multiple crystallographic and cryo-EM studies over the past 10 years have revealed important insight into the Mtr4 structure and interactions with protein and nucleic acid binding partners. These structures place Mtr4 at the center of a dynamic process that recruits RNA substrates and presents them to the exosome. In this review, we summarize the available Mtr4 structures and highlight gaps in our current understanding.

The Impact of Alcohol Hangover on Simulated Driving Performance During a 'Commute to Work'-Zero and Residual Alcohol Effects Compared.

Driving is increasing across the world and road traffic accidents are a major cause of serious injuries and fatalities. The link between alcohol consumption and impaired driving has long been established and has led to legislation in many countries, with enforcement of legal limits based on blood alcohol concentration levels. Alcohol hangover research is an emerging field with a range of laboratory and naturalistic studies now clearly demonstrating the significant impairments that can result from hangover, even when alcohol levels are measured at or close to zero the day following a social drinking occasion. Driving is a commonplace activity but requires competency with a range of complex and potentially demanding tasks. Driving impaired can have serious consequences, including death and serious injury. There have been only limited alcohol hangover driving studies. The studies presented examined the consequences of alcohol hangover with a driving simulator contrasting a group with zero residual alcohol (N = 26) next day and another with residual alcohol (N = 26) assessed with breathalyzer in the morning before undertaking a 20 min commute to work. All participants completed a morning drive after a night without alcohol consumption and another after a night of social drinking. The driving scenarios were relatively demanding including traffic and pedestrians, traffic lights and other potential hazards in a mixed rural and urban journey. Subjective hangover and workload were assessed in addition to a range of driving performance variables, including divided attention, steering control and driving violations. Analyses contrasted driving in the no alcohol condition with the residual alcohol condition. The combined groups data (N = 52) was contrasted with the zero and residual alcohol groups. Significant contrasts were found for a range of driving measures, including divided attention, vehicle control, and driving violations as well as perceived workload. The pattern of impairment was broadly similar across both groups, indicating that whether or not residual alcohol was present, consistent driving impairment was seen. The relatively high number of significant variables may reflect the increased cognitive demand of the 20 min commute drive including busy and complex urban environments. This was also reflected in the significant increase in perceived workload recorded across the 6 dimensions of the National Aeronautics and Space Administration Task Load Index (NASA-TLX). Associations between subjective measures and driving performance with hangover suggested a potential lack of awareness of impairment, though were limited in number. The overall findings indicate that the levels of impairment seen reflect those seen with alcohol impaired driving, even when breath alcohol is zero.

Closing in on ATPase Activity by an RNA Helicase.

In this issue of Structure, Absmeier et al. (2020) describe the molecular mechanisms employed by an RNA helicase to prevent premature ATP hydrolysis upon nucleotide binding.

Sensitivity to Experiencing Alcohol Hangovers: Reconsideration of the 0.11% Blood Alcohol Concentration (BAC) Threshold for Having a Hangover.

The 2010 Alcohol Hangover Research Group consensus paper defined a cutoff blood alcohol concentration (BAC) of 0.11% as a toxicological threshold indicating that sufficient alcohol had been consumed to develop a hangover. The cutoff was based on previous research and applied mostly in studies comprising student samples. Previously, we showed that sensitivity to hangovers depends on (estimated) BAC during acute intoxication, with a greater percentage of drinkers reporting hangovers at higher BAC levels. However, a substantial number of participants also reported hangovers at comparatively lower BAC levels. This calls the suitability of the 0.11% threshold into question. Recent research has shown that subjective intoxication, i.e., the level of severity of reported drunkenness, and not BAC, is the most important determinant of hangover severity. Non-student samples often have a much lower alcohol intake compared to student samples, and overall BACs often remain below 0.11%. Despite these lower BACs, many non-student participants report having a hangover, especially when their subjective intoxication levels are high. This may be the case when alcohol consumption on the drinking occasion that results in a hangover significantly exceeds their "normal" drinking level, irrespective of whether they meet the 0.11% threshold in any of these conditions. Whereas consumers may have relative tolerance to the adverse effects at their "regular" drinking level, considerably higher alcohol intake-irrespective of the absolute amount-may consequentially result in a next-day hangover. Taken together, these findings suggest that the 0.11% threshold value as a criterion for having a hangover should be abandoned.

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation.

Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.

A YopH PTP1B Chimera Shows the Importance of the WPD-Loop Sequence to the Activity, Structure, and Dynamics of Protein Tyrosine Phosphatases.

To study factors that affect WPD-loop motion in protein tyrosine phosphatases (PTPs), a chimera of PTP1B and YopH was created by transposing the WPD loop from PTP1B to YopH. Several subsequent mutations proved to be necessary to obtain a soluble, active enzyme. That chimera, termed chimera 3, retains productive WPD-loop motions and general acid catalysis with a pH dependency similar to that of the native enzymes. Kinetic isotope effects show the mechanism and transition state for phosphoryl transfer are unaltered. Catalysis of the chimera is slower than that of either of its parent enzymes, although its rate is comparable to those of most native PTPs. X-ray crystallography and nuclear magnetic resonance were used to probe the structure and dynamics of chimera 3. The chimera's structure was found to sample an unproductive hyper-open conformation of its WPD loop, a geometry that has not been observed in either of the parents or in other native PTPs. The reduced catalytic rate is attributed to the protein's sampling of this conformation in solution, reducing the fraction in the catalytically productive loop-closed conformation.