Comprehensive analysis of CXXX sequence space reveals that Saccharomyces cerevisiae GGTase-I mainly relies on a2X substrate determinants.

Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a 3-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C-cysteine, a-aliphatic, L-often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving 2 distinct reporters were employed in this study to assess Saccharomyces cerevisiae GGTase-I specificity, for which limited data exist, toward all 8,000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of noncanonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a nonpolar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity.

Evaluating protein prenylation of human and viral CaaX sequences using a humanized yeast system.

Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections. Prenylated proteins with a C-terminal CaaX sequence are targeted by CaaX-type prenyltransferases and proteases. To aid investigations of these enzymes and their targets, we developed Saccharomyces cerevisiae strains that express these human enzymes instead of their yeast counterparts. These strains were developed in part to explore human prenyltransferase specificity because of findings that yeast FTase has expanded specificity for sequences deviating from the CaaX consensus (i.e. atypical sequence and length). The humanized yeast strains displayed robust prenyltransferase activity against CaaX sequences derived from human and pathogen proteins containing typical and atypical CaaX sequences. The system also recapitulated prenylation of heterologously expressed human proteins (i.e. HRas and DNAJA2). These results reveal that substrate specificity is conserved for yeast and human farnesyltransferases but is less conserved for type I geranylgeranyltransferases. These yeast systems can be easily adapted for investigating the prenylomes of other organisms and are valuable new tools for helping define the human prenylome, which includes physiologically important proteins for which the CaaX modification status is unknown.

Comprehensive analysis of CXXX sequence space reveals that S. cerevisiae GGTase-I mainly relies on a2X substrate determinants.

Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a three-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C - cysteine, a - aliphatic, L - often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving two distinct reporters were employed in this study to assess S. cerevisiae GGTase-I specificity, for which limited data exists, towards all 8000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of non-canonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a non-polar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity.

A Humanized Yeast System for Evaluating the Protein Prenylation of a Wide Range of Human and Viral CaaX Sequences.

The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur in 1-2% of proteins within yeast and human proteomes. Recently, non-canonical CaaX sequences in addition to shorter and longer length CaX and CaaaX sequences have been identified that can be prenylated. Much of the characterization of prenyltransferases has relied on the yeast system because of its genetic tractability and availability of reporter proteins, such as the a-factor mating pheromone, Ras GTPase, and Ydj1 Hsp40 chaperone. To compare the properties of yeast and human prenyltransferases, including the recently expanded target specificity of yeast farnesyltransferase, we have developed yeast strains that express human farnesyltransferase or geranylgeranyltransferase-I in lieu of their yeast counterparts. The humanized yeast strains display robust prenyltransferase activity that functionally replaces yeast prenyltransferase activity in a wide array of tests, including the prenylation of a wide variety of canonical and non-canonical human CaaX sequences, virus encoded CaaX sequences, non-canonical length sequences, and heterologously expressed human proteins HRas and DNAJA2. These results reveal highly overlapping substrate specificity for yeast and human farnesyltransferase, and mostly overlapping substrate specificity for GGTase-I. This yeast system is a valuable tool for further defining the prenylome of humans and other organisms, identifying proteins for which prenylation status has not yet been determined.

A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences.

The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: cysteine-aliphatic1-aliphatic2-variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high-throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.

A difference-in-difference approach to examining the impact of cannabis legalization on disparities in the use of cigarettes and cannabis in the United States, 2004-17.

AIMS: To estimate the impact of recreational and medical cannabis laws (RCL, MCL) on the use of cannabis and cigarettes in the United States. DESIGN: A difference-in-difference approach was applied to data from the 2004-17 National Survey on Drug Use and Health (NSDUH). SETTING: United States. PARTICIPANTS: Nationally representative cross-sectional survey of Americans aged 12 years and older (combined analytical sample for 2004-17, n = 783 663). MEASUREMENTS: Data on past-month use of (1) cigarettes and (2) cannabis were used to classify respondents into four groups: cigarette and cannabis co-use, cigarette-only use, cannabis-only use or no cigarette or cannabis use. State of residence was measured by self-report. MCL/RCL status came from state government websites. FINDINGS: Difference-in-difference analyses suggest that MCL was associated with an increase in cigarette-cannabis co-use overall [adjusted odds ratio (aOR) = 1.09; 95% confidence interval (CI) = 1.02-1.16], with the greatest increases among those aged 50 years and above (aOR = 1.60; CI = 1.39-1.84), married (aOR = 1.19; CI = 1.07-1.31), non-Hispanic (NH) black (aOR = 1.14; CI = 1.02-1.07) and with a college degree or above (aOR = 1.15; CI = 1.06-1.24). MCL was associated with increases in cigarette-only use among those aged 50 years and above (aOR = 1.07; CI = 1.01-1.14) and NH black (aOR = 1.16; CI = 1.06-1.27) and increases in cannabis-only use among those aged 50 years and above (aOR = 1.24; CI = 1.07-1.44) and widowed/divorced/separated (aOR = 1.18; CI = 1.01-1.37). RCL was associated with an increase in cannabis-only use overall (aOR = 1.21; 95% CI = 1.09-1.34), a decline in cigarette-only use overall (aOR = 0.89; 95% CI = 0.81-0.97) and increases in co-use among those who were married (aOR = 1.24; CI = 1.02-1.50) and aged 50 years and above (aOR = 1.37; CI = 1.03-1.84). CONCLUSIONS: Recreational and medical cannabis legalization have had a varying impact on the use, and co-use, of cannabis and cigarettes in the United States.

Trends in cannabis use among adults with children in the home in the United States, 2004-2017: impact of state-level legalization for recreational and medical use.

BACKGROUND AND AIMS: Cannabis use among parents may be increasing with legalization, but perception of associated risk has declined. The study investigated the association between cannabis legalization and cannabis use among adults with children in the home over time in the United States (US). DESIGN: A difference-in-difference approach was applied to public and restricted-use data from the 2004-2017 National Survey on Drug Use and Health (NSDUH), an annual cross-sectional survey. SETTING: A representative sample of the United States. PARTICIPANTS/CASES: Respondents ages 18+ with children living in the home drawn from the NSDUH (n = 287,624), which is administered to non-institutionalized civilians in the 50 states and District of Columbia. MEASUREMENTS: Exposures were year and state-level cannabis policy in state of residence annually. Outcomes were past-30-day cannabis use and daily cannabis use. Sociodemographic variables included age, gender, marital status, annual family income, race/ethnicity, educational attainment, and strength of state-level tobacco control. FINDINGS: In 2017, past-month cannabis use (11.9%, 9.3%, and 6.1%) and daily cannabis use (4.2%, 3.2%, and 2.3%) were more common in states with recreational marijuana laws (RML), followed by states with medical marijuana laws (MML) and without legal cannabis use, respectively. RML and MML were associated with significantly higher prevalence of past-month cannabis use (adjusted odds ratio [AOR] = 1.28, 95% confidence interval [CI] = 1.12-1.46; AOR = 1.12, 95% CI = 1.03-1.22) and daily cannabis use (AOR = 1.25, 95% CI = 1.03-1.51; AOR = 1.16, 95% CI = 1.02-1.32), respectively. The impact of MML was particularly salient among adults ages 50+ and the highest income and education subgroups. CONCLUSIONS: Among adults with children living in the home, cannabis use appears to be more common in US states with legalized cannabis use compared with states with no legal cannabis use. Recreational legalization appears to increase use among adults with children in the home broadly across nearly all sociodemographic groups, whereas the effect of legalization for medical use is heterogeneous by age and socioeconomic status.

Impact of state-level cannabis legalization on poly use of alcohol and cannabis in the United States, 2004-2017.

BACKGROUND: Cannabis policy has shifted toward legalization in many parts of the United States (US). While attention has been focused on whether legalization will lead to changes in cannabis use, it is conceivable that legalization will also affect use of substances that individuals frequently use with cannabis. This study assessed whether cannabis legalization impacted the prevalence of poly use of cannabis and alcohol from 2004 to 2017 and estimated the prevalence of cannabis and alcohol poly use in 2017. METHODS: Public and restricted-use data from the US 2004-2017 National Survey on Drug Use and Health were analyzed. Data on past-month cannabis and alcohol use were assessed each year. Cannabis legalization was determined by the presence or absence of medical marijuana laws (MML) and recreational marijuana laws (RML) in each state. Difference-in-difference approach was used to estimate the association of MML and RML on cannabis and alcohol use overall and by sociodemographic subgroups (e.g., age, income, education). RESULTS: Between 2004 and 2017, poly use of cannabis and alcohol increased while alcohol-only use decreased. MML were associated with increases in poly use only among adults over age 50 and among those with higher annual incomes and higher education, while RML were associated with increases in poly use broadly among adults across sociodemographic groups. CONCLUSIONS: Legalization of cannabis was associated with increases in cannabis-alcohol poly use in the US. RML were associated with increases across demographics, while the impact of MML was more limited to certain sociodemographic groups.

Cannabis use among US adults with anxiety from 2008 to 2017: The role of state-level cannabis legalization.

BACKGROUND: Cannabis use is more common among adults with anxiety. Cannabis legalization is occurring rapidly across the United States (US) and individuals may use cannabis to cope with anxiety. This study investigated whether cannabis use across the US has changed differentially by anxiety status and by state cannabis legalization for medical (MML) and/or recreational use (RML). METHODS: Public and restricted-use data from the 2004 to 2017 National Survey on Drug Use and Health, an annual cross-sectional, nationally representative survey of US individuals, were analyzed. The prevalence of past-30-day cannabis use by anxiety status in 2017 was estimated among respondents ages ≥18 (n = 42,554) by sociodemographics and state-level cannabis law. Weighted logistic regressions with continuous year as the predictor for the linear time trend were used to examine the time trends in cannabis use by anxiety and cannabis law status from 2004 to 2017 (total combined analytic sample n = 398,967). RESULTS: Cannabis use was consistently two to three times higher among those with high anxiety compared to those with some or no anxiety and was higher in states with RML compared to MML or no MML/RML. Cannabis use has increased over time among those with and without anxiety overall, in MML states, and in states without MML/RML; with a faster increase in cannabis use among those with high anxiety compared to lower anxiety in states with MML. CONCLUSIONS: Cannabis use is increasing among American adults overall, yet is disproportionately common among Americans with anxiety especially among those residing in states where cannabis has been legalized.

In-transition culture of experimentation with cannabis in Latin American college students: A new role within a potential drug use sequencing pattern.

INTRODUCTION AND AIMS: Given a scenario of intense discussion about the legal situation of cannabis users worldwide, this paper aims to investigate the role of cannabis within a drug use sequencing pattern. DESIGN AND METHODS: Data came from a representative sample of college students from 27 Brazilian capitals (n = 12 711). We analysed the patterns of transition from/to the first use of cannabis to/from the first use of alcohol, tobacco and seven other illegal drugs. Survival analysis methods were used to analyse age of onset data on all potential drug pairs. Drugs that were not specified as the target drug pair tested in each survival model were included as time-varying covariates in all models. RESULTS: We found significant transitions from alcohol [adjusted hazard ratio (aHR) = 1.41, 95% confidence interval (CI) 1.15-1.73, P < 0.001] and inhalants (aHR = 1.56, 95% CI 1.26-1.93, P < 0.001) to cannabis. Moreover, we found significant transitions from cannabis to alcohol (aHR = 2.40, 95% CI 1.47-3.91, P < 0.001), cocaine (aHR = 7.47, 95% CI = 4.26-13.09, P < 0.001), prescription opioids (aHR = 2.16, 95% CI 1.29-3.63, P < 0.01) and tranquilisers (aHR = 1.51, 95% CI 1.11-2.06, P < 0.01). DISCUSSION AND CONCLUSIONS: Overall, our findings point to a strategic role of cannabis within drug first use sequence pattern. We had an important and unexpected finding-the bi-directional relationship between alcohol and cannabis. In addition, the first use of cannabis still precedes the first use of cocaine and non-medical use of tranquilisers and prescription opioids. [Castaldelli-Maia JM, Nicastri S, Cerdá M, Kim JH, Oliveira LG, Andrade AG, Martins, SS. In-transition culture of experimentation with cannabis in Latin American college students: A new role within a potential drug use sequencing pattern. Drug Alcohol Rev 2017;00:000-000].

State-level medical marijuana laws, marijuana use and perceived availability of marijuana among the general U.S. population.

BACKGROUND: Little is known on how perceived availability of marijuana is associated with medical marijuana laws. We examined the relationship between medical marijuana laws (MML) and the prevalence of past-month marijuana use, with perceived availability of marijuana. METHODS: Data were from respondents included in the National Survey of Drug Use and Health restricted use data portal 2004-2013. Multilevel logistic regression of individual-level data was used to test differences between MML and non-MML states and changes in prevalence of past-month marijuana use and perceived availability from before to after passage of MML among adolescents, young adults and older adults controlling for demographics. RESULTS: Among adults 26+, past-month prevalence of marijuana use increased from 5.87% to 7.15% after MML passage (Adjusted Odds Ratio (AOR): 1.24 [1.16-1.31]), but no change in prevalence of use was found for 12-17 or 18-25 year-olds. Perceived availability of marijuana increased after MML was enacted among those 26+ but not in younger groups. Among all age groups, prevalence of marijuana use and perception of it being easily available was higher in states that would eventually pass MML by 2013 compared to those that had not. Perceived availability was significantly associated with increased risk of past-month marijuana use in all age groups. CONCLUSION: Evidence suggests perceived availability as a driver of change in use of marijuana due to MML. To date, this has only occurred in adults 26+ and different scenarios that could explain this change need to be further explored.

A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent phenotypes.

The modifications occurring to CaaX proteins have largely been established using few reporter molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, and COOH-terminal methylation. Here, we investigated the coupling of these modifications in the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is resistant to post-isoprenylation events. These results expand on the complexity of protein isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of Ydj1p and perhaps other CaaX proteins.

Older, Less Regulated Medical Marijuana Programs Have Much Greater Enrollment Rates Than Newer 'Medicalized' Programs.

Twenty-three states and the District of Columbia have passed laws implementing medical marijuana programs. The nineteen programs that were in operation as of October 2014 collectively had over one million participants. All states (including D.C.) with medical marijuana laws require physicians directly or indirectly to authorize the use of marijuana at their discretion, yet little is known about how medical marijuana programs vary regarding adherence to basic principles of medical practice and associated rates of enrollment. To explore this, we analyzed marijuana programs according to seven components of traditional medical care and pharmaceutical regulation. We then examined enrollment rates, while controlling for potentially confounding state characteristics. We found that fourteen of the twenty-four programs were nonmedical and collectively enrolled 99.4 percent of participants nationwide, with enrollment rates twenty times greater than programs deemed to be "medicalized." Policy makers implementing or amending medical marijuana programs should consider the powerful relationship between less regulation and greater enrollment. Researchers should consider variations across programs when assessing programs' population-level effects.

Symptoms of alcohol dependence and smoking initiation and persistence: a longitudinal study among US adults.

BACKGROUND: A large number of adults report symptoms of, but do not meet diagnostic criteria for, an alcohol use disorder. Yet, little is known about the relationship between symptoms of alcohol use disorders and the initiation and persistence of smoking. This study prospectively examines the relationship between having 1-2 symptoms of alcohol dependence (without abuse) and smoking initiation and persistence as well as nicotine dependence over a 3-year period among adults in the United States. METHODS: Data were drawn from Wave 1 (2001-2002) and Wave 2 (2004-2005) of the National Epidemiologic Survey on Alcohol and Related Conditions. Relationships between Wave 1 symptoms of alcohol dependence, alcohol abuse, and alcohol dependence and initiation and persistence of cigarette smoking and nicotine dependence at Wave 2 were examined using logistic regression analyses. Analyses were adjusted for demographics, mood and anxiety disorders. RESULTS: Symptoms of alcohol dependence were associated with smoking initiation at Wave 2. There was no association between symptoms of alcohol dependence and smoking persistence. Symptoms of alcohol dependence predicted incident and persistent nicotine dependence. Findings persisted after adjusting for demographic characteristics and mood/anxiety disorders. CONCLUSIONS: Even 1-2 symptoms of alcohol dependence are associated with increased vulnerability to smoking initiation and onset and persistence of nicotine dependence at a similar strength as alcohol use disorders. Efforts at smoking cessation must address problematic alcohol use, even at the subclinical level, in order to improve efficacy.

Real-time MR imaging-guided laser atrial septal puncture in swine.

PURPOSE: The authors performed this study to report their initial preclinical experience with real-time magnetic resonance (MR) imaging-guided atrial septal puncture by using a MR imaging-conspicuous blunt laser catheter that perforates only when energized. MATERIALS AND METHODS: The authors customized a 0.9-mm clinical excimer laser catheter with a receiver coil to impart MR imaging visibility at 1.5 T. Seven swine underwent laser transseptal puncture under real-time MR imaging. MR imaging signal-to-noise ratio profiles of the device were obtained in vitro. Tissue traversal force was tested with a calibrated meter. Position was corroborated with pressure measurements, oximetry, angiography, and necropsy. Intentional non-target perforation simulated serious complication. RESULTS: Embedded MR imaging antennae accurately reflected the position of the laser catheter tip and profile in vitro and in vivo. Despite having an increased profile from the microcoil, the 0.9-mm laser catheter traversed in vitro targets with similar force (0.22 N +/- 0.03) compared with the unmodified laser. Laser puncture of the atrial septum was successful and accurate in all animals. The laser was activated an average of 3.8 seconds +/- 0.4 before traversal. There were no sequelae after 6 hours of observation. Necropsy revealed 0.9-mm holes in the fossa ovalis in all animals. Intentional perforation of the aorta and atrial free wall was evident immediately. CONCLUSIONS: MR imaging-guided laser puncture of the interatrial septum is feasible in swine and offers controlled delivery of perforation energy by using an otherwise blunt catheter. Instantaneous soft tissue imaging provides immediate feedback on safety.