Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway

Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.ope

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 90 (HSP90) inhibitors have emerged as a promising class of anti-cancer drugs in both solid and hematologic malignancies. The HSP90 family includes the cytosolic HSP90 (HSP90AA1), the ER paralogue gp96 (HSP90B1) and the mitochondrial member TRAP1 (HSP90L). We evaluated the <it>in vitro </it>anti-tumor activity and mechanism of action of PU-H71, a novel purine scaffold HSP90 inhibitor in human multiple myeloma cell lines.</p> <p>Methods</p> <p>Multiple human myeloma cell lines including cells that are resistant to corticosteroids and bortezimab were treated with PU-H71, followed by analysis of cell viability, cell cycle progression and apoptosis, by flow cytometry and caspase 3 immunoblot. Induction of unfolded protein response was studied by XBP-1 s immunoblot. The role of gp96 was further assessed by small hairpin RNA knockdown of gp96 before treatment with PU-H71.</p> <p>Results</p> <p>PU-H71 has potent <it>in vitro </it>anti-myeloma activity in both drug-sensitive and drug-resistant cell lines. PU-H71 activates the unfolded protein response and induces caspase-dependent apoptosis. The stable gp96 knockdown human myeloma cell line was found to be more resistant to PU-H71 and other HSP90 inhibitors including 17-AAG and 17-DMAG, even though these cells are more sensitive to conventional anti-myeloma drugs.</p> <p>Conclusion</p> <p>We conclude that PU-H71 is a promising drug for the treatment of myeloma. Our finding further suggests that PU-H71 and the geldanamycin analogues work in part by inhibiting the endoplasmic reticulum gp96 along with the cytosolic HSP90.</p

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

Supporting genetics in primary care: investigating how theory can inform professional education

Evidence indicates that many barriers exist to the integration of genetic case finding into primary care. We conducted an exploratory study of the determinants of three specific behaviours related to using breast cancer genetics referral guidelines effectively: 'taking a family history', 'making a risk assessment', and 'making a referral decision'. We developed vignettes of primary care consultations with hypothetical patients, representing a wide range of genetic risk for which different referral decisions would be appropriate. We used the Theory of Planned Behavior to develop a survey instrument to capture data on behavioural intention and its predictors (attitude, subjective norm, and perceived behavioural control) for each of the three behaviours and mailed it to a sample of Canadian family physicians. We used correlation and regression analyses to explore the relationships between predictor and dependent variables. The response rate was 96/125 (77%). The predictor variables explained 38-83% of the variance in intention across the three behaviours. Family physicians' intentions were lower for 'making a risk assessment' (perceived as the most difficult) than for the other two behaviours. We illustrate how understanding psychological factors salient to behaviour can be used to tailor professional educational interventions; for example, considering the approach of behavioural rehearsal to improve confidence in skills (perceived behavioural control), or vicarious reinforcement as where participants are sceptical that genetics is consistent with their role (subjective norm)

Valproate, a Mood Stabilizer, Induces WFS1 Expression and Modulates Its Interaction with ER Stress Protein GRP94

Valproate is a standard treatment for bipolar disorder and a first-line mood stabilizer. The molecular mechanisms underlying its actions in bipolar disorder are unclear. It has been suggested that the action of valproate is linked to changes in gene expression and induction of endoplasmic reticulum (ER) stress-response proteins.Here we show that valproate modulates the ER stress response through the regulation of WFS1, an important component for mitigating ER stress. Therapeutic concentrations of valproate induce expression of WFS1 mRNA and activate the WFS1 promoter. In addition, WFS1 forms a complex with GRP94, an ER stress-response protein, in which valproate dose-dependently enhances its dissociation from GRP94.These results suggest that the therapeutic effects of valproate in bipolar disorder may be mediated by WFS1 expression and its dissociation from GRP94

Identification of the PDI-Family Member ERp90 as an Interaction Partner of ERFAD

In the endoplasmic reticulum (ER), members of the protein disulfide isomerase (PDI) family perform critical functions during protein maturation. Herein, we identify the previously uncharacterized PDI-family member ERp90. In cultured human cells, we find ERp90 to be a soluble ER-luminal glycoprotein that comprises five potential thioredoxin (Trx)-like domains. Mature ERp90 contains 10 cysteine residues, of which at least some form intramolecular disulfides. While none of the Trx domains contain a canonical Cys-Xaa-Xaa-Cys active-site motif, other conserved cysteines could endow the protein with redox activity. Importantly, we show that ERp90 co-immunoprecipitates with ERFAD, a flavoprotein involved in ER-associated degradation (ERAD), through what is most likely a direct interaction. We propose that the function of ERp90 is related to substrate recruitment or delivery to the ERAD retrotranslocation machinery by ERFAD

Interfering with Glycolysis Causes Sir2-Dependent Hyper-Recombination of Saccharomyces cerevisiae Plasmids

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key metabolic regulator implicated in a variety of cellular processes. It functions as a glycolytic enzyme, a protein kinase, and a metabolic switch under oxidative stress. Its enzymatic inactivation causes a major shift in the primary carbohydrate flux. Furthermore, the protein is implicated in regulating transcription, ER-to-Golgi transport, and apoptosis. We found that Saccharomyces cerevisiae cells null for all GAPDH paralogues (Tdh1, Tdh2, and Tdh3) survived the counter-selection of a GAPDH–encoding plasmid when the NAD+ metabolizing deacetylase Sir2 was overexpressed. This phenotype required a fully functional copy of SIR2 and resulted from hyper-recombination between S. cerevisiae plasmids. In the wild-type background, GAPDH overexpression increased the plasmid recombination rate in a growth-condition dependent manner. We conclude that GAPDH influences yeast episome stability via Sir2 and propose a model for the interplay of Sir2, GAPDH, and the glycolytic flux

Assessing Perceived Risk and STI Prevention Behavior: A National Population-Based Study with Special Reference to HPV

Aim: This thesis aims to provide a multidimensional assessment of infection risks and to evaluate strategies for HPV prevention including vaccination with quadrivalent HPVvaccines, dose-level vaccine effectiveness and condom use in high STI risk situations. Methods: Multiple population-based registers and questionnaire responses provided data for this thesis. Various multivariable and univariate regression models were fit. Findings: Overall, quadrivalent HPV-vaccination was highly effective against genital warts (GW) also referred to as condyloma, which is the first HPV disease endpoint possible to measure. However, effectiveness was contingent upon young age-at-first vaccination, with effectiveness declining steadily the older the age-at-first vaccination. Among women above 20 years of age there was low to immeasurable effectiveness and suggestive evidence vaccinations in this age group tended to reach women at high GW risk. There were marked socioeconomic disparities in the opportunistic (on-demand with co-pay) vaccination strategy evaluated, with women and girls who have parents with the highest education level compared to the lowest having a 15 times greater likelihood to be vaccinated (Study III). Once vaccination was initiated, however, high parental education level was unrelated to vaccination completion. Maximum protection against GW was found among girls vaccinated under the age of 17 who had received three doses of the vaccine. No differences in effectiveness were found for girls who received twodoses between ages 10-16 with that of those who received three-doses between ages 17- 19 (Study IV). GW affects more men than women in Sweden as of 2010 with 453 per 100 000 men and 365 per 100 000 women treated. A decline between 25-30% was seen between 2006 and 2010 among women in the age groups with the highest vaccination coverage. No decline was found amongst men and their GW incidence has steadily increased between 2006 and 2010 (Study II). Reported condom use in high risk situations was low among both men and women, with 41% of men and 34% of women reporting always/almost always condom use with temporary partners. STI risk perception was also low, with approximately 10% of sexually active respondents considering themselves at large risk of contracting an STI. There was no association between men’s condom use and their STI risk perception but there was an association for women (Study I). Conclusions: Results suggest that males bear a substantial burden of HPV-related condyloma where incidence has dropped among women. When planning HPVvaccination among females, efforts should target girls under age 14 for maximum effectiveness. Quadrivalent HPV-vaccination offers most protection against condyloma at three doses. Gross social inequity was found with opportunistic HPV-vaccination. There were large gender differences in factors associated with condom use in high risk situations and STI risk perceptions