Polyglutamine-mediated ribotoxicity disrupts proteostasis and stress responses in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat in the Huntingtin (HTT) gene, encoding a homopolymeric polyglutamine (polyQ) tract. Although mutant HTT (mHTT) protein is known to aggregate, the links between aggregation and neurotoxicity remain unclear. Here we show that both translation and aggregation of wild-type HTT and mHTT are regulated by a stress-responsive upstream open reading frame and that polyQ expansions cause abortive translation termination and release of truncated, aggregation-prone mHTT fragments. Notably, we find that mHTT depletes translation elongation factor eIF5A in brains of symptomatic HD mice and cultured HD cells, leading to pervasive ribosome pausing and collisions. Loss of eIF5A disrupts homeostatic controls and impairs recovery from acute stress. Importantly, drugs that inhibit translation initiation reduce premature termination and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.

A hierarchical assembly pathway directs the unique subunit arrangement of TRiC/CCT.

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.

Modulating environmental signals to reveal mechanisms and vulnerabilities of cancer persisters.

Cancer persister cells are able to survive otherwise lethal doses of drugs through nongenetic mechanisms, which can lead to cancer regrowth and drug resistance. The broad spectrum of molecular differences observed between persisters and their treatment-naïve counterparts makes it challenging to identify causal mechanisms underlying persistence. Here, we modulate environmental signals to identify cellular mechanisms that promote the emergence of persisters and to pinpoint actionable vulnerabilities that eliminate them. We found that interferon-γ (IFNγ) can induce a pro-persistence signal that can be specifically eliminated by inhibition of type I protein arginine methyltransferase (PRMT) (PRMTi). Mechanistic investigation revealed that signal transducer and activator of transcription 1 (STAT1) is a key component connecting IFNγ's pro-persistence and PRMTi's antipersistence effects, suggesting a previously unknown application of PRMTi to target persisters in settings with high STAT1 expression. Modulating environmental signals can accelerate the identification of mechanisms that promote and eliminate cancer persistence.

The cumulative incidence of cisplatin-induced hearing loss in young children is higher and develops at an early stage during therapy compared with older children based on 2052 audiological assessments.

BACKGROUND: Ototoxicity is a common adverse event of cisplatin treatment. The authors investigated the development of cisplatin-induced hearing loss (CIHL) over time in children with cancer by age and examined the influence of other clinical characteristics on the course of CIHL. METHODS: Data from Canadian patients with childhood cancer were retrospectively reviewed. Hearing loss was graded according to International Society of Pediatric Oncology criteria. The Kaplan-Meier method was applied to estimate the cumulative incidence of CIHL for the total cohort and according to age. Cox regression models were used to explore the effects of independent variables on CIHL development up to 3 years after the start of therapy. RESULTS: In total, 368 patients with 2052 audiological assessments were included. Three years after initiating therapy, the cumulative incidence of CIHL was highest in patients aged ≤5 years (75%; 95% confidence interval [CI], 66%-84%), with a rapid increase observed to 27% (95% CI, 21%-35%) at 3 months and to 61% (95% CI, 53%-69%) at 1 year, compared with patients aged >5 years (48%; 95% CI, 37%-62%; P < .001). The total cumulative dose of cisplatin at 3 months (per 100 mg/m2 increase: hazard ratio [HR], 1.20; 95% CI, 1.01-1.41) vincristine (HR, 2.87; 95% CI, 1.89-4.36) and the total duration of concomitantly administered antibiotics (>30 days: HR, 1.85; 95% CI, 1.17-2.95) further influenced CIHL development over time. CONCLUSIONS: In young children, the cumulative incidence of CIHL is higher compared with that in older children and develops early during therapy. The course of CIHL is further influenced by the total cumulative dose of cisplatin and other ototoxic (co-)medication. These results highlight the need for audiological monitoring at each cisplatin cycle.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns.

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

A targeted covalent small molecule inhibitor of HIV-1 fusion.

We describe a low molecular weight covalent inhibitor targeting a conserved lysine residue within the hydrophobic pocket of HIV-1 glycoprotein-41. The inhibitor bound selectively to the hydrophobic pocket and exhibited an order of magnitude enhancement of anti-fusion activity against HIV-1 compared to its non-covalent counterpart. The findings represent a significant advance in the quest to obtain non-peptide fusion inhibitors.

Phosphorylation at distinct subcellular locations underlies specificity in mTORC2-mediated activation of SGK1 and Akt.

mTORC2 lies at the intersection of signaling pathways that control metabolism and ion transport through phosphorylation of the AGC-family kinases, the Akt and SGK1 proteins. How mTORC2 targets these functionally distinct downstream effectors in a context-specific manner is not known. Here, we show that the salt- and blood pressure-regulatory hormone, angiotensin II (AngII) stimulates selective mTORC2-dependent phosphorylation of SGK1 (S422) but not Akt (S473 and equivalent sites). Conventional PKC (cPKC), a critical mediator of the angiotensin type I receptor (AT1R, also known as AGTR1) signaling, regulates the subcellular localization of SIN1 (also known as MAPKAP1) and SGK1. Inhibition of cPKC catalytic activity disturbs SIN1 and SGK1 subcellular localization, re-localizing them from the nucleus and a perinuclear compartment to the plasma membrane in advance of hormonal stimulation. Surprisingly, pre-targeting of SIN1 and SGK1 to the plasma membrane prevents SGK1 S422 but not Akt S473 phosphorylation. Additionally, we identify three sites on SIN1 (S128, S315 and S356) that are phosphorylated in response to cPKC activation. Collectively, these data demonstrate that SGK1 activation occurs at a distinct subcellular compartment from that of Akt and suggests a mechanism for the selective activation of these functionally distinct mTORC2 targets through subcellular partitioning of mTORC2 activity.

Evaluating Canadians' Values for Drug Coverage Decision Making.

BACKGROUND: Decision makers are facing growing challenges in prioritizing drugs for reimbursement because of soaring drug costs and increasing pressures on financial resources. In addition to cost and effectiveness, payers are using other values to dictate which drugs are prioritized for funding, yet there are limited data on the Canadian public's priorities. OBJECTIVES: To measure the relative societal importance of values considered most relevant in informing drug reimbursement decisions in a representative sample of Canadians. METHODS: An online survey of 2539 Canadians aged 19 years and older was performed in which 13 values used in drug funding prioritization were ranked and then weighted using an analytic hierarchy process. RESULTS: Canadians value safe and efficacious drugs that have certainty of evidence. The values ranked in the top 5 by most of our subjects were potential effect on quality of life (65.4%), severity of the disease (62.6%), ability of drug to work (61.1%), safety (60.5%), and potential to extend life (49.4%). Values related to patient or disease characteristics such as rarity, socioeconomic status, and health and lifestyle choices held the lowest rankings and weights. CONCLUSIONS: Canadians value, above all, treatment-related factors (eg, efficacy and safety) and disease-related factors (eg, severity and equity). Decision makers are currently using additional justifications to prioritize drugs for reimbursement, such as rarity and unmet need, which were not found to be highly valued by Canadians. Decision makers should integrate the public's values into a Canadian reimbursement framework for prioritization of drugs competing for limited funds.

Chemically induced vesiculation as a platform for studying TMEM16F activity.

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: determining phosphorylation sites in peptides with multiple serines.

Trivalent boron species readily react with protonated phosphopeptides to give addition products with the loss of boron ligands. In the present study, trimethoxyborane (TMB), diisopropoxymethylborane (DIPM), and diethylmethoxyborane (DEMB) were allowed to react with four phosphopeptides, VsSF, LSsF, LsGASA, and VSGAsA (lower-case s indicates phosphoserine). Each of the phosphopeptides contains one serine that is phosphorylated and one that is not. Under collision-activated dissociation (CAD) conditions, the boron-derivatized peptides give fragmentation patterns that differ significantly from that of the protonated phosphopeptide. The patterns vary, depending on the number of labile (i.e., alkoxy) ligands on the boron. In general, boron derivatization increases the yield of phosphate-containing sequence ions, but dramatic effects are only seen with certain reagent/peptide combinations. However, the suite of reagents provides a means of altering and increasing the information content of phosphopeptide CAD spectra.