TORC1 is an essential regulator of nutrient-controlled proliferation and differentiation in Leishmania.

Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite proliferation and differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8, and identify new parasite-specific proteins that interact in this complex. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient-rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites are unable to respond to nutrients to differentiate into proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. We additionally show that RPTOR1-/- metacyclic promastigotes develop into amastigotes but do not proliferate in the mammalian host to cause pathology. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.

Cysteine post-translational modifications regulate protein interactions of caveolin-3.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Essential roles for deubiquitination in Leishmania life cycle progression.

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.

"A Woman in A Man's World": A Pilot Qualitative Study of Challenges Faced by Women Veterans During and After Deployment.

Women Veterans face gender-specific challenges to military life and post-deployment readjustment, including gender-based discrimination and military sexual trauma. Despite recent military initiatives to address these issues, women still experience unique challenges during military service. This study examines spontaneous comments about gender-specific challenges to military life that were made by participants in a qualitative study of women's transitions to civilian life after deployment to Iraq or Afghanistan. METHODS: Women Veterans who were enrolled at a New England VA hospital and who had deployed to the U.S. conflicts in Iraq and Afghanistan participated in this qualitative study (N = 22). Interview queries and initial coding structure were developed through an extensive literature review. An iterative coding process generated additional themes identified in the data. For this project, codes regarding self-initiated reports of gender-specific challenges that fell outside the scope of the study's initial interview agenda were reviewed for thematic analysis. RESULTS: The following three self-initiated themes emerged among 12 respondents: 1) gender-based scrutiny and discrimination; 2) the military's inadequate position and response to military sexual trauma; and 3) disadvantages to women service members living in a male-dominated environment. Across all three themes emerged a sub-theme in which women perceived their unique needs to be inconvenient and/or disregarded. Respondents described how these challenges disrupted their lives during and after military service. CONCLUSION: Results imply gender-specific challenges and military sexual trauma remain critical concerns for women Veterans well after deployment had ended, and that improved policy may have long-term health implications.

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.

Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation.

Leishmania mexicana has a large family of cyclin-dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2-related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co-expression of a CRK3 transgene during rapamycin-induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3(T178E) mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.

Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen.

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

Oral health care for underserved children in the United States.

Dental caries is the most common chronic disease of childhood with approximately 25% of children from low-income families entering kindergarten without ever having seen a dentist ( Larsen, Larsen, Handwerker, Kim, & Rosenthal, 2009 ). Youth, poverty, and race are characteristics of populations susceptible to oral disease (Dye, Xianfen, & Thorton-Evans, 2012). Services delivering oral health care to underserved populations are referred to as dental safety-net clinics. This article explores the impact of the dental safety-net on improving access to oral health care for underserved children in the United States.

Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.

Long-term consequences of membership in trajectory groups of delinquent behavior in an urban sample: violence, drug use, interpersonal, and neighborhood attributes.

Research on stability and change in delinquent behavior over time has important implications for both the individual and the criminal justice system. The present research looks at this issue by examining the associations between the trajectories of delinquent behavior in adolescence and adult functioning. Data for the present study are from a four-wave longitudinal study of African American and Hispanic participants. Participants provided information at mean ages 14, 19, 24, and 29. We used growth mixture modeling to extract trajectory groups of delinquent behavior in adolescence and young adulthood. Regression analyses were conducted to examine whether memberships in the trajectory groups of delinquent behavior from mean age 14 to mean age 24 were associated with violence, substance abuse and dependence, partner discord, peer substance use, and residence in a high-crime neighborhood at mean age 29 when compared with the reference trajectory group of participants with low or no delinquent behavior. Four trajectory groups of delinquent behavior were identified: the no/low, the decreasing, the moderate, and the high persistent trajectory groups. Memberships in the trajectory groups were significantly correlated with variations in adult functioning. Memberships in some trajectory groups of delinquent behavior are significant predictors of later violent behavior, substance abuse and dependence, partner discord, peer substance use, and residence in a high-crime neighborhood. The findings reinforce the importance of investing in interventions to address different patterns of delinquent behavior. Findings are discussed in relation to previous investigations with non-Hispanic White samples.

Palmitoylation regulates the magnitude of HCN4-mediated currents in mammalian cells.

The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.

Biomarkers of exposure and potential harm in exclusive users of electronic cigarettes and current, former, and never smokers.

Electronic cigarette (EC) aerosol emissions generally contain fewer and lower concentrations of harmful and potentially harmful constituents, compared with cigarette smoke. Further studies are needed to establish whether decreased emissions translate to reduced health risks for EC users. In a cross-sectional study, biomarkers of exposure (BoE) to certain tobacco smoke toxicants and biomarkers of potential harm (BoPH), associated with biological processes linked to the potential development of smoking-related diseases and oxidative stress, were assessed in solus Vuse ECs users and current, former, and never smokers. In total, 213 participants were enrolled, and smoking status was confirmed by urinary cotinine, exhaled carbon monoxide, and N-(2-cyanoethyl)valine levels (EC users and former smokers only). During confinement participants used their usual product (EC or cigarette) as normal and BoE and BoPHs were assessed via blood, 24-h urine, and physiological assessment. Significantly lower levels of all urinary BoE; MHBMA, HMPMA, 3-HPMA, NNN, 3-OH-B[a]P, S-PMA, NNAL (all p < 0.0001), and TNeq (p = 0.0074) were observed in EC users when compared with smokers. Moreover, significantly lower levels were observed in EC users for 3 of the 7 BoPH measured, carboxyhaemoglobin (p < 0.0001), soluble intercellular adhesion molecule-1 (p = 0.0028), and 11-dehydrothromboxane B2 (p = 0.0012), when compared with smokers. As compared with smokers, solus Vuse EC users have significantly lower exposure to tobacco toxicants for the BoE, and 3 BoPH measured. These results add to the weight of evidence supporting EC as part of a tobacco harm reduction strategy.

Comorbid trajectories of tobacco and marijuana use as related to psychological outcomes.

Heterogeneous classes of comorbid trajectories of tobacco and marijuana use were examined in order to determine how they are related to subsequent antisocial behavior, poor self-control, and internalizing behavior. Data are from a 4-wave longitudinal study of African American (n = 243) and Puerto Rican (n = 232) adolescents and adults in the community. Logistic regression analyses were employed to measure the association between the comorbid trajectories of tobacco and marijuana use and the psychological difficulty variables. The authors found 6 joint trajectory groups. The authors compared the non-or-experimental tobacco/marijuana use group with the other user groups in each of the psychological difficulty domains. The infrequent tobacco/late-onset marijuana use and chronic tobacco/marijuana use groups differed most strongly from the non-or-experimental tobacco/marijuana use group across the antisocial behavior, poor self-control, and internalizing problems domains. The chronic tobacco/maturing out marijuana use group also had significant associations in each of these domains. The infrequent tobacco/marijuana use and late-onset tobacco/infrequent marijuana use groups had no or weak associations with the psychological outcomes. Tobacco and marijuana cessation programs should identify and address comorbid use of tobacco and marijuana, and antisocial behavior, poor self-control, and internalizing problems, which are associated with histories of comorbid use of the 2 substances.

Individuality and contextual influences on drug dependence: a 15-year Prospective longitudinal study of adolescents from Harlem.

In this 15-year longitudinal study the authors investigated individual and contextual factors that predispose adolescents from a disadvantaged urban area to drug dependence in adulthood. Adolescents were recruited from schools serving East Harlem in New York City. Of the 838 participants followed to adulthood, 59% were women, 55% were African American, and 45% were Puerto Rican. Self-report data were obtained on externalizing and internalizing problems, substance use, and contextual influences across adolescence and young adulthood. Drug dependence was assessed in adulthood. Multivariate logistic regressions of drug dependence were performed on the whole sample and separately by gender. Each of the domains was associated with adult drug dependence. Although mean gender differences were found, most associations of risk factors with drug dependence did not vary significantly by gender. Treating externalizing and internalizing problems, reducing substance use, and providing coping skills for adverse contextual influences in adolescence and young adulthood may reduce the likelihood of becoming drug dependent in adulthood.

Palmitoylation regulates cellular distribution of and transmembrane Ca flux through TrpM7.

The bifunctional cation channel/kinase TrpM7 is ubiquitously expressed and regulates embryonic development and pathogenesis of several common diseases. The TrpM7 integral membrane ion channel domain regulates transmembrane movement of divalent cations, and its kinase domain controls gene expression via histone phosphorylation. Mechanisms regulating TrpM7 are elusive. It exists in two populations in the cell: at the cell surface where it controls divalent cation fluxes, and in intracellular vesicles where it controls zinc uptake and release. Here we report that TrpM7 is palmitoylated at a cluster of cysteines at the C terminal end of its Trp domain. Palmitoylation controls the exit of TrpM7 from the endoplasmic reticulum and the distribution of TrpM7 between cell surface and intracellular pools. Using the Retention Using Selective Hooks (RUSH) system, we demonstrate that palmitoylated TrpM7 traffics from the Golgi to the surface membrane whereas non-palmitoylated TrpM7 is sequestered in intracellular vesicles. We identify the Golgi-resident enzyme zDHHC17 and surface membrane-resident enzyme zDHHC5 as responsible for palmitoylating TrpM7 and find that TrpM7-mediated transmembrane calcium uptake is significantly reduced when TrpM7 is not palmitoylated. The closely related channel/kinase TrpM6 is also palmitoylated on the C terminal side of its Trp domain. Our findings demonstrate that palmitoylation controls ion channel activity of TrpM7 and that TrpM7 trafficking is dependant on its palmitoylation. We define a new mechanism for post translational modification and regulation of TrpM7 and other Trps.

ATR Inhibitor AZD6738 (Ceralasertib) Exerts Antitumor Activity as a Monotherapy and in Combination with Chemotherapy and the PARP Inhibitor Olaparib.

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2-M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent. SIGNIFICANCE: This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

Developmental trajectories of marijuana use from adolescence to adulthood: personality and social role outcomes.

Longitudinal trajectories of marijuana use from adolescence into adulthood were examined for adverse life-course outcomes among African-Americans and Puerto Ricans. Data for marijuana use were analyzed at four points in time and on participants' personality attributes, work functioning, and partner relations in adulthood using growth mixture modeling. Each of the three marijuana-use trajectory groups (maturing-out, late-onset, and chronic marijuana-users) had greater adverse life-course outcomes than a nonuse or low-use trajectory group. The chronic marijuana-use trajectory group was highly associated with criminal behavior and partners' marijuana use in adulthood. Treatment programs for marijuana use should also directly address common adverse life-course outcomes users may already be experiencing.

Course of comorbidity of tobacco and marijuana use: psychosocial risk factors.

INTRODUCTION: This longitudinal study examined the psychosocial factors associated with the comorbidity of pairs of tobacco and marijuana use trajectories from adolescence extending into adulthood in two ethnic groups, Blacks and Puerto Ricans. METHODS: Data on psychosocial functioning and tobacco and marijuana use at four points in time were obtained. RESULTS: The association between the trajectories of tobacco and marijuana use was quite high. Pairs of comorbid trajectories of tobacco and marijuana use may share at least three kinds of influence: (a) a constellation of externalizing personality risk factors, (b) Depressive Mood and low Ego Integration, and (c) identification with certain group values. DISCUSSION: Knowledge of the risk and protective factors for pairs of comorbid trajectories of use may strengthen the foundation for individual and group targets for prevention and treatment programs.

Control of protein palmitoylation by regulating substrate recruitment to a zDHHC-protein acyltransferase.

Although palmitoylation regulates numerous cellular processes, as yet efforts to manipulate this post-translational modification for therapeutic gain have proved unsuccessful. The Na-pump accessory sub-unit phospholemman (PLM) is palmitoylated by zDHHC5. Here, we show that PLM palmitoylation is facilitated by recruitment of the Na-pump α sub-unit to a specific site on zDHHC5 that contains a juxtamembrane amphipathic helix. Site-specific palmitoylation and GlcNAcylation of this helix increased binding between the Na-pump and zDHHC5, promoting PLM palmitoylation. In contrast, disruption of the zDHHC5-Na-pump interaction with a cell penetrating peptide reduced PLM palmitoylation. Our results suggest that by manipulating the recruitment of specific substrates to particular zDHHC-palmitoyl acyl transferases, the palmitoylation status of individual proteins can be selectively altered, thus opening the door to the development of molecular modulators of protein palmitoylation for the treatment of disease.