Factors associated with physical function among people with systemic sclerosis: a SPIN cohort cross-sectional study.

OBJECTIVES: To compare physical function in systemic sclerosis (SSc, scleroderma) to general population normative data and identify associated factors. METHODS: Scleroderma Patient-centered Intervention Network Cohort participants completed the Physical Function domain of the Patient-Reported Outcomes Measurement Information System Version 2 upon enrolment. Multivariable linear regression was used to assess associations of sociodemographic, lifestyle, and disease-related variables. RESULTS: Among 2,385 participants, mean physical function T-score (43.7, SD = 8.9) was ∼2/3 of a standard deviation (SD) below the US general population (mean = 50, SD = 10). Factors associated in multivariable analysis included older age (-0.74 points per SD years, 95% CI -0.78 to -1.08), female sex (-1.35, -2.37 to -0.34), fewer years of education (-0.41 points per SD in years, -0.75 to -0.07), being single, divorced, or widowed (-0.76, -1.48 to -0.03), smoking (-3.14, -4.42 to -1.85), alcohol consumption (0.79 points per SD drinks per week, 0.45-1.14), BMI (-1.41 points per SD, -1.75 to -1.07), diffuse subtype (-1.43, -2.23 to -0.62), gastrointestinal involvement (-2.58, -3.53 to -1.62), digital ulcers (-1.96, -2.94 to -0.98), moderate (-1.94, -2.94 to -0.93) and severe (-1.76, -3.24 to -0.28) small joint contractures, moderate (-2.10, -3.44 to -0.76) and severe (-2.54, -4.64 to -0.44) large joint contractures, interstitial lung disease (-1.52, -2.27 to -0.77), pulmonary arterial hypertension (-3.72, -4.91 to -2.52), rheumatoid arthritis (-2.10, -3.64 to -0.56) and idiopathic inflammatory myositis (-2.10, -3.63 to -0.56). CONCLUSION: Physical function is impaired for many individuals with SSc and associated with multiple disease factors.

Structural Basis of PML-RARA Oncoprotein Targeting by Arsenic Unravels a Cysteine Rheostat Controlling PML Body Assembly and Function.

PML nuclear bodies (NB) are disrupted in PML-RARA-driven acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) cures 70% of patients with APL, driving PML-RARA degradation and NB reformation. In non-APL cells, arsenic binding onto PML also amplifies NB formation. Yet, the actual molecular mechanism(s) involved remain(s) elusive. Here, we establish that PML NBs display some features of liquid-liquid phase separation and that ATO induces a gel-like transition. PML B-box-2 structure reveals an alpha helix driving B2 trimerization and positioning a cysteine trio to form an ideal arsenic-binding pocket. Altering either of the latter impedes ATO-driven NB assembly, PML sumoylation, and PML-RARA degradation, mechanistically explaining clinical ATO resistance. This B2 trimer and the C213 trio create an oxidation-sensitive rheostat that controls PML NB assembly dynamics and downstream signaling in both basal state and during stress response. These findings identify the structural basis for arsenic targeting of PML that could pave the way to novel cancer drugs. SIGNIFICANCE: Arsenic curative effects in APL rely on PML targeting. We report a PML B-box-2 structure that drives trimer assembly, positioning a cysteine trio to form an arsenic-binding pocket, which is disrupted in resistant patients. Identification of this ROS-sensitive triad controlling PML dynamics and functions could yield novel drugs. See related commentary by Salomoni, p. 2505. This article is featured in Selected Articles from This Issue, p. 2489.

Exploration of nuclear body-enhanced sumoylation reveals that PML represses 2-cell features of embryonic stem cells.

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml-/- mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.

Retinoids in hematology: a timely revival?

The retinoic acid receptors (RARA, RARB, and RARG) are ligand-regulated nuclear receptors that act as transcriptional switches. These master genes drew significant interest in the 1990s because of their key roles in embryogenesis and involvement in a rare malignancy, acute promyelocytic leukemia (APL), in which the RARA (and very rarely, RARG or RARB) genes are rearranged, underscoring the central role of deregulated retinoid signaling in leukemogenesis. Several recent provocative observations have revived interest in the roles of retinoids in non-APL acute myeloid leukemia (AML), as well as in normal hematopoietic differentiation. We review the role of retinoids in hematopoiesis, as well as in the treatment of non-APL AMLs. From this perspective, broader uses of retinoids in the management of hematopoietic tumors are discussed.

Suicidal ideation and attempt in adolescents exposed to maternal smoking across pregnancy and childhood: A 20-year prospective cohort study.

BACKGROUND: Few studies investigated the association between maternal smoking during pregnancy and offspring suicide risk, none considering postnatal smoking exposure. We investigated associations between maternal smoking patterns during the pre- and postnatal periods and adolescent suicidal ideation and attempt. METHODS: We identified longitudinal patterns of maternal smoking from the prenatal period to the end of childhood (children's age 12 years, 10 assessments) among participants in the Québec Longitudinal Study of Child Development (N = 1623). We estimated associations between maternal smoking patterns and offspring self-reported suicidal ideation and attempt (ages 13-20). Background confounding factors (e.g., socioeconomic, familial, mental health) were controlled using propensity score inverse-probability weighting (IPW). RESULTS: Participants reporting suicidal ideation and attempt were 9.3% and 8.4%, respectively. We identified four maternal smoking patterns: non-smoking (66.5%), increasing (5.5%), decreasing (9.3%), persistent (18.5%). Children exposed to persistent (OR=2.92, CI=1.99-4.30) and increasing (OR=2.06, CI=1.13-3.74) maternal smoking were more likely to attempt suicide, compared to non-exposed children. Accounting for confounding factors using IPW fully explained the association between increasing smoking and suicide attempt (OR=0.95, CI=0.39-2.09) but only reduced the association between persistent exposure and suicide attempt (OR=2.30, CI=1.04-4.99). No increased suicide attempt risk was found for children of mothers with a decreased smoking pattern. We found no associations for suicidal ideation. LIMITATIONS: Propensity score cannot account for unmeasured confounding factors; attrition limits generalizability. CONCLUSIONS: Offspring of mothers who smoked persistently and heavily prenatally and postnatally were at increased risk of suicide attempt in adolescence. Future studies should elucidate biological and psychosocial mechanisms potentially at play in these associations.

A genetically informed study on the association of cannabis, alcohol, and tobacco smoking with suicide attempt.

Use of substances such as cannabis, alcohol, and tobacco, has been associated with increased risk of suicide attempt in several observational studies. However, establishing whether these associations are causal is challenging when using observational designs. To evaluate the potential causal contributions of cannabis use, alcohol use, and tobacco smoking to suicide attempt, we applied two-sample Mendelian randomization, an instrumental variable approach using single-nucleotide polymorphisms (SNPs) as instrumental variables for three exposures: lifetime cannabis use (yes/no; 42 instrument SNPs; GWAS sample size [N] = 162,082), alcohol use (drinks-per-week; 53 instrument SNPs; N = 941,280), and tobacco smoking (initiation, yes/no; 156 instrument SNPs; N = 1,232,091; heaviness; 27 instrument SNPs; N = 337,334). The main outcome was suicide attempt measured from hospital records (N = 50,264). All data come from publicly available summary statistics of genome-wide association studies of participants of European ancestry. We found evidence supporting a possible causal role of cannabis (OR = 1.18; 95% CI = 1.01-1.37, P = 0.032), alcohol (OR = 1.95; 95% CI = 1.15-3.32, P = 0.013), and smoking (initiation, OR = 1.90; 95% CI = 1.54-2.34, P < 0.001; heaviness, OR = 2.13; 95% CI = 1.13-3.99; P = 0.019) on suicide attempt. Using multivariable Mendelian randomization, we found that only cannabis showed a direct pathway to suicide attempt (P = 0.001), suggesting that the effect of alcohol and smoking was mediated by the other substance use phenotypes. No evidence was found for reverse causation, i.e., associations of suicide attempt on cannabis (P = 0.483), alcohol (P = 0.234), smoking initiation (P = 0.144), and heaviness (P = 0.601). In conclusion, evidence from this quasi-experimental study based on genetic data from large-scale GWASs are consistent with a causal role of cannabis, alcohol, and tobacco smoking on suicide attempt.

Cannabis use, depression and suicidal ideation in adolescence: direction of associations in a population based cohort.

BACKGROUND: To clarify the direction of the association between frequency of cannabis use, depressive symptoms and suicidal ideation from 15 to 20 years using cross-lagged analyses. METHOD: We included 1606 adolescents from the province of Québec followed since 1997 with information on frequency of cannabis use (none/monthly/weekly), depression (defined as being in the top 10% symptoms) and serious suicidal ideation at ages 15, 17 and 20 years. RESULTS: The prevalence of weekly cannabis use increased from 7.0% at age 15 years to 15.6% by age 20 years. Adolescents who reported using cannabis weekly at one age were 11 to 15 times more likely to continue using cannabis over time. In longitudinal cross-lagged analyses, weekly cannabis use at age 15 was associated with greater odds (OR=2.19, 95% CI=1.04-4.58) of suicidal ideation two years later. However, other substance use (alcohol, tobacco, other drugs) fully explained this association. Further, depression predicted subsequent weekly cannabis use, even after adjusting for comorbid other substance use (eg, for depression at 15 years predicting cannabis use at 17 years: OR=2.30, 95% CI=1.19-4.43). LIMITATIONS: Quantity of cannabis consumed was not measured. CONCLUSION: Findings suggest that depressive symptoms in adolescence may represent a risk factor for weekly cannabis consumption, which once initiated is likely to remain chronic. Weekly cannabis use increased risk for suicidal ideation, but not independently from other substance use including alcohol, tobacco and other drugs.

Classic and Variants APLs, as Viewed from a Therapy Response.

Most acute promyelocytic leukemia (APL) are caused by PML-RARA, a translocation-driven fusion oncoprotein discovered three decades ago. Over the years, several other types of rare X-RARA fusions have been described, while recently, oncogenic fusion proteins involving other retinoic acid receptors (RARB or RARG) have been associated to very rare cases of acute promyelocytic leukemia. PML-RARA driven pathogenesis and the molecular basis for therapy response have been the focus of many studies, which have now converged into an integrated physio-pathological model. The latter is well supported by clinical and molecular studies on patients, making APL one of the rare hematological disorder cured by targeted therapies. Here we review recent data on APL-like diseases not driven by the PML-RARA fusion and discuss these in view of current understanding of "classic" APL pathogenesis and therapy response.

In-utero and perinatal influences on suicide risk: a systematic review and meta-analysis.

BACKGROUND: Adverse in-utero and perinatal conditions might contribute to an increased suicide risk throughout the lifespan; however, existing evidence is sparse and contradictory. We aimed to investigate in-utero and perinatal exposures associated with suicide, suicide attempt, and suicidal ideation. METHODS: We did a systematic review and meta-analysis and searched MEDLINE, Embase, and PsycINFO from inception to Jan 24, 2019, for population-based prospective studies that investigated the association between in-utero and perinatal factors and suicide, suicide attempt, and suicidal ideation. Only papers published in English in peer-reviewed journals were considered. Two researchers independently extracted formal information (eg, country, year, duration of follow-up) and number of cases and non-cases exposed and non-exposed to each risk factor. We calculated pooled odds ratios (ORs) with 95% CIs using random-effects models and used meta-regression to investigate heterogeneity. This study was registered with PROSPERO, number CRD42018091205. FINDINGS: We identified 42 eligible studies; they had a low risk of bias (median quality score 9/9 [IQR 8-9]). Family or parental characteristics, such as high birth order (eg, for fourth-born or later-born vs first-born, pooled OR 1·51 [95% CIs 1·21-1·88]), teenage mothers (1·80 [1·52-2·14]), single mothers (1·57 [1·31-1·89]); indices of socioeconomic position, such as low maternal (1·36 [1·28-1·46]) and paternal (1·38 [1·27-1·51]) education; and fetal growth (eg, low birthweight 1·30 [1·09-1·55] and small for gestational age 1·18 [1·00-1·40]) were associated with higher suicide risk. Father's age, low gestational age, obstetric characteristics (eg, caesarean section), and condition or exposure during pregnancy (eg, maternal smoking or hypertensive disease) were not associated with higher suicide risk. Similar patterns of associations were observed for suicide attempt and suicidal ideation; however, these results were based on a lower number of studies. In meta-regression, differences in length of follow-up explained most between-study heterogeneity (inital I2 ranged from 0 to 79·5). INTERPRETATION: These findings suggest that prenatal and perinatal characteristics are associated with increased suicide risk during the life course, supporting the developmental origin of health and diseases hypothesis for suicide. The low number of studies for some risk factors, especially for suicide attempt and ideation, leaves gaps in knowledge that need to be addressed. The mechanisms underlying the reported associations and their causal nature still remain unclear. FUNDING: Horizon 2020 (EU).

Diurnal cortisol and mental well-being in middle and older age: evidence from four cohort studies.

OBJECTIVES: We conducted an individual participant meta-analysis to test the hypothesis that cortisol patterns indicative of dysregulated hypothalamic-pituitary-adrenal axis functioning would be prospectively associated with poorer well-being at follow-up. SETTING: Four large UK-based cohort studies. PARTICIPANTS: Those providing valid salivary or serum cortisol samples (n=7515 for morning cortisol; n=1612 for cortisol awakening response) at baseline (age 44-82) and well-being data on the Warwick Edinburgh Mental Wellbeing Scale at follow-up (0-8 years) were included. RESULTS: Well-being was not associated with morning cortisol, diurnal slope or awakening response though a borderline association with evening cortisol was found. Adjusting for sex and follow-up time, each 1 SD increase in evening cortisol was associated with a -0.47 (95% CI -1.00 to 0.05) point lower well-being. This was attenuated by adjustment for body mass index, smoking and socioeconomic position. Between-study heterogeneity was low. CONCLUSIONS: This study does not support the hypothesis that diurnal cortisol is prospectively associated with well-being up to 8 years later. However, replication in prospective studies with cortisol samples over multiple days is required.

MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus.

UNLABELLED: Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction. IMPORTANCE: We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.

Formation of Nup98-containing nuclear bodies in HeLa sublines is linked to genomic rearrangements affecting chromosome 11.

Nup98 is an important component of the nuclear pore complex (NPC) and also a rare but recurrent target for chromosomal translocation in leukaemogenesis. Nup98 contains multiple cohesive Gly-Leu-Phe-Gly (GLFG) repeats that are critical notably for the formation of intranuclear GLFG bodies. Previous studies have reported the existence of GLFG bodies in cells overexpressing exogenous Nup98 or in a HeLa subline (HeLa-C) expressing an unusual elevated amount of endogenous Nup98. Here, we have analysed the presence of Nup98-containing bodies in several human cell lines. We found that HEp-2, another HeLa subline, contains GLFG bodies that are distinct from those identified in HeLa-C. Rapid amplification of cDNA ends (RACE) revealed that HEp-2 cells express additional truncated forms of Nup98 fused to a non-coding region of chromosome 11q22.1. Cytogenetic analyses using FISH and array-CGH further revealed chromosomal rearrangements that were distinct from those observed in leukaemic cells. Indeed, HEp-2 cells feature a massive amplification of juxtaposed NUP98 and 11q22.1 loci on a chromosome marker derived from chromosome 3. Unexpectedly, minor co-amplifications of NUP98 and 11q22.1 loci were also observed in other HeLa sublines, but on rearranged chromosomes 11. Altogether, this study reveals that distinct genomic rearrangements affecting NUP98 are associated with the formation of GLFG bodies in specific HeLa sublines.

25-Hydroxyvitamin D and cognitive performance in mid-life.

Hypovitaminosis D has been linked with poor cognitive function, particularly in older adults, but studies lack a lifespan approach; hence, the effects of reverse causality remain unknown. In the present study, we aimed to assess the relationship between 25-hydroxyvitamin D (25(OH)D) concentrations and subsequent cognitive performance in mid-adulthood and the influence of earlier life factors, including childhood cognitive ability, on this association. Information for the present study was obtained from the members of the 1958 British birth cohort (n 6496). Serum 25(OH)D concentration, indicating vitamin D status, was measured at age 45 years. Verbal memory (immediate and delayed word recall), verbal fluency (animal naming) and speed of processing were tested at age 50 years. Information on childhood cognitive ability, educational attainment, vitamin D-related behaviours and other covariates was collected prospectively from participants throughout their life. Childhood cognitive ability and educational attainment by age 42 years were strongly correlated with cognitive performance at age 50 years and with several vitamin D-related behaviours in mid-adulthood, but not with 25(OH)D concentrations at age 45 years. Participants with both low (<25 nmol/l) and high (≥75 nmol/l) 25(OH)D concentrations at age 45 years performed significantly worse on immediate word recall. The associations attenuated after adjustment for childhood cognitive ability, education, and socio-economic position; however, for the immediate word recall test, there was a non-linear association with 25(OH)D after further adjustment for obesity, menopausal status, smoking, alcohol consumption, physical activity and depressive symptoms at age 45 years (P(curvature)=0·01). The present study demonstrated that 25(OH)D concentrations were non-linearly associated with immediate word recall in mid-life. A clarification of the level of 25(OH)D concentrations that is most beneficial for predicting better cognitive performance in mid-life is required.

Role of promyelocytic leukemia protein in host antiviral defense.

Several pathways have been implicated in the establishment of antiviral state in response to interferon (IFN), one of which implicates the promyelocytic leukemia (PML) protein. The PML gene has been discovered 20 years ago and has led to new insights into oncogenesis, apoptosis, cell senescence, and antiviral defense. PML is induced by IFN, leading to a marked increase of expression of PML isoforms and the number of PML nuclear bodies (NBs). PML is the organizer of the NBs that contains at least 2 permanent NB-associated proteins, the IFN-stimulated gene product Speckled protein of 100 kDa (Sp100) and death-associated dead protein (Daxx), as well as numerous other transient proteins recruited in these structures in response to different stimuli. Accumulating reports have implicated PML in host antiviral defense and revealed various strategies developed by viruses to disrupt PML NBs. This review will focus on the regulation of PML and the implication of PML NBs in conferring resistance to DNA and RNA viruses. The role of PML in mediating an IFN-induced antiviral state will also be discussed.

Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies.

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.

HSP90 protein stabilizes unloaded argonaute complexes and microscopic P-bodies in human cells.

Key components of the miRNA-mediated gene regulation pathway are localized in cytoplasmic processing bodies (P-bodies). Mounting evidence suggests that the presence of microscopic P-bodies are not always required for miRNA-mediated gene regulation. Here we have shown that geldanamycin, a well-characterized HSP90 inhibitor, abolishes P-bodies and significantly reduces Argonaute and GW182 protein levels but does not affect the miRNA level and the efficiency of miRNA-mediated gene repression; however, it significantly impairs siRNA loading and the efficacy of exogenous siRNA. Our data suggests that HSP90 protein chaperones Argonautes before binding RNA and may facilitate efficient loading of small RNA.

Characterization of SENP7, a SUMO-2/3-specific isopeptidase.

The modification of proteins by SUMO (small ubiquitin-related modifier) plays important roles in regulating the activity, stability and cellular localization of target proteins. Similar to ubiquitination, SUMO modification is a dynamic process that can be reversed by SENPs [SUMO-1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidases]. To date, six SENPs have been discovered in humans, although knowledge of their regulation, specificity and biological functions is limited. In the present study, we report that SENP7 has a restricted substrate specificity, being unable to process SUMO precursors and displaying paralogue-specific isopeptidase activity. The C-terminal catalytic domain of SENP7 efficiently depolymerized poly-SUMO-2 chains but had undetectable activity against poly-SUMO-1 chains. SENP7 also displayed isopeptidase activity against di-SUMO-2- and SUMO-2-modified RanGAP1 (Ran GTPase-activating protein 1) but had limited activity against SUMO-1-modified RanGAP1. in vivo, full-length SENP7 was localized to the nucleoplasm and preferentially reduced the accumulation of high-molecular-mass conjugates of SUMO-2 and SUMO-3 compared with SUMO-1. Small interfering RNA-mediated ablation of SENP7 expression led to the accumulation of high-molecular-mass SUMO-2 species and to the accumulation of promyelocytic leukaemia protein in subnuclear bodies. These findings suggest that SENP7 acts as a SUMO-2/3-specific protease that is likely to regulate the metabolism of poly-SUMO-2/3 rather than SUMO-1 conjugation in vivo.

RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation.

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

Impact of the interaction between herpes simplex virus type 1 regulatory protein ICP0 and ubiquitin-specific protease USP7 on activation of adeno-associated virus type 2 rep gene expression.

Expression of the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 in transfected cells reactivates rep gene expression from integrated adeno-associated virus (AAV) type 2 genomes via a mechanism that requires both its RING finger and USP7 interaction domains. In this study, we found that the rep reactivation defect of USP7-binding-negative ICP0 mutants can be overcome by further deletion of sequences in the C-terminal domain of ICP0, indicating that binding of USP7 to ICP0 is not directly required. Unlike the case in transfected cells, only the RING finger domain of ICP0 was essential for rep gene reactivation during HSV-1 infection. However, mutants unable to bind to USP7 activate HSV-1 gene expression and reactivate rep gene expression with reduced efficiencies. These results further elucidate the role of ICP0 as a helper factor for AAV replication and illustrate that care is required when extrapolating from the properties of ICP0 in transfection assays to events occurring during HSV-1 infection.

Helper functions required for wild type and recombinant adeno-associated virus growth.

The human parvovirus Adeno-Associated virus (AAV-2) has been classified as a Dependovirus because it requires the presence of a helper virus to achieve a productive replication cycle. Several viruses such as Adenovirus (Ad), Herpes Simplex Virus (HSV), Vaccinia virus, and human papillomaviruses (HPV) can provide the helper activities required for AAV growth. The studies on the helper activities provided by adenovirus have provided useful information not only to understand the AAV-2 biology but also to develop tools for the production of recombinant AAV particles (rAAV). This review will focus on the current knowledge about the helper activities provided by the most extensively studied helper viruses, Ad and HSV-1, and also illustrate the methods used to supply the helper functions rAAV assembly.