Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Loading of the ring-shaped Mcm2-7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2-7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2-7 complexes. Loading of each Mcm2-7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2-7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.

Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level.

Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2-7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2-7 loading specificity and that Mcm2-7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2-7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes.

Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain.

Viral hijacking of cellular processes relies on the ability to mimic the structure or function of cellular proteins. Many viruses encode ubiquitin ligases to facilitate infection, although the mechanisms by which they select their substrates are often unknown. The Herpes Simplex Virus type-1-encoded E3 ubiquitin ligase, ICP0, promotes infection through degradation of cellular proteins, including the DNA damage response E3 ligases RNF8 and RNF168. Here we describe a mechanism by which this viral E3 hijacks a cellular phosphorylation-based targeting strategy to degrade RNF8. By mimicking a cellular phosphosite, ICP0 binds RNF8 via the RNF8 forkhead associated (FHA) domain. Phosphorylation of ICP0 T67 by CK1 recruits RNF8 for degradation and thereby promotes viral transcription, replication, and progeny production. We demonstrate that this mechanism may constitute a broader viral strategy to target other cellular factors, highlighting the importance of this region of the ICP0 protein in countering intrinsic antiviral defenses.

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV's ability to mobilize the host actin cytoskeleton within the cell's nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell's nucleus, and yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

Elevated homocysteine is negatively correlated with plasma cystathionine ß-synthase activity in givosiran-treated patients.

Givosiran is a subcutaneously administered, liver-targeted RNA interference (RNAi) therapeutic that has been approved for treating acute hepatic porphyria (AHP). Elevation in plasma homocysteine (hyperhomocysteinemia) has been reported in AHP patients, and treatment with givosiran has been reported to further increase homocysteine levels in some patients. The mechanism of homocysteine elevation during givosiran treatment is unknown, but has been hypothesized to be mediated by a reduction in activity of cystathionine ß-synthase (CBS), which uses homocysteine as a substrate. A liquid chromatography-tandem mass spectrometry-based assay was adapted to measure circulating CBS activity. Using plasma collected from the Phase III ENVISION study, CBS activity was measured to directly evaluate whether it is associated with elevated homocysteine levels in givosiran-treated patients. CBS activity was reduced following givosiran treatment and both homocysteine and methionine levels were inversely correlated with CBS activity. Following administration of a supplement containing vitamin B6, a cofactor for CBS, in four patients during the trial, plasma CBS activity was found to increase, mirroring a corresponding decrease in homocysteine levels. These results support the hypothesis that elevated homocysteine levels following givosiran treatment result from a reduction of CBS activity and that vitamin B6 supplementation lowers homocysteine levels by increasing CBS activity.

Treatment response and neurofilament light chain levels with long-term patisiran in hereditary transthyretin-mediated amyloidosis with polyneuropathy: 24-month results of an open-label extension study.

BACKGROUND: Longitudinal changes in neurofilament light chain (NfL) levels were evaluated alongside prespecified clinical assessments 24 months into the patisiran Global open-label extension (OLE) study in patients with ATTRv amyloidosis with polyneuropathy. METHODS: All patients enrolled in the Global OLE, from phase III APOLLO and phase II OLE parent studies, received patisiran. Assessments included measures of polyneuropathy (modified Neuropathy Impairment Score+7 (mNIS+7)), quality of life (QOL; Norfolk QOL-Diabetic Neuropathy questionnaire (Norfolk QOL-DN)), and plasma NfL. RESULTS: Patients receiving patisiran in the parent study (APOLLO-patisiran, n = 137; phase II OLE-patisiran, n = 25) demonstrated sustained improvements in mNIS+7 (mean change from parent study baseline (95% confidence interval): APOLLO-patisiran -4.8 (-8.9, -0.6); phase II OLE-patisiran -5.8 (-10.5, -1.2)) and Norfolk QOL-DN (APOLLO-patisiran -2.4 (-7.2, 2.3)), and maintained reduced NfL levels at Global OLE 24 months. After initiating patisiran in the Global OLE, APOLLO-placebo patients (n = 49) demonstrated stabilized mNIS+7, improved Norfolk QOL-DN, and significantly reduced NfL levels. Patisiran continued to demonstrate an acceptable safety profile. Earlier patisiran initiation was associated with a lower exposure-adjusted mortality rate. CONCLUSIONS: Long-term patisiran treatment led to sustained improvements in neuropathy and QOL, with NfL demonstrating potential as a biomarker for disease progression and treatment response in ATTRv amyloidosis with polyneuropathy.

Activin E is a TGFß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming Growth Factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, similar to ActC. Collectively, our results establish ActE as an ALK7 ligand, thereby providing a link between genetic and in vivo studies of ActE as a regulator of adipose tissue.

Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity.

Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C, encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease.

Widespread haploid-biased gene expression enables sperm-level natural selection.

Sperm are haploid but must be functionally equivalent to distribute alleles equally among progeny. Accordingly, gene products are shared through spermatid cytoplasmic bridges that erase phenotypic differences between individual haploid sperm. Here, we show that a large class of mammalian genes are not completely shared across these bridges. We call these genes "genoinformative markers" (GIMs) and show that a subset can act as selfish genetic elements that spread alleles unevenly through murine, bovine, and human populations. We identify evolutionary pressure to avoid conflict between sperm and somatic function as GIMs are enriched for testis-specific gene expression, paralogs, and isoforms. Therefore, GIMs and sperm-level natural selection may help to explain why testis gene expression patterns are an outlier relative to all other tissues.

Neurofilament Light Chain as a Biomarker of Hereditary Transthyretin-Mediated Amyloidosis.

OBJECTIVE: To identify changes in the proteome associated with onset and progression of hereditary transthyretin-mediated (hATTR) amyloidosis, also known as ATTRv amyloidosis, we performed an observational, case-controlled study that compared proteomes of patients with ATTRv amyloidosis and healthy controls. METHODS: Plasma levels of >1,000 proteins were measured in patients with ATTRv amyloidosis with polyneuropathy who received either placebo or patisiran in a Phase 3 study of patisiran (APOLLO), and in healthy controls. The effect of patisiran on the time profile of each protein was determined by linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) was further assessed with an orthogonal quantitative approach. RESULTS: Levels of 66 proteins were significantly changed with patisiran vs placebo, with NfL change most significant (p < 10-20). Analysis of changes in protein levels demonstrated that the proteome of patients treated with patisiran trended toward that of healthy controls at 18 months. Healthy controls' NfL levels were 4-fold lower than in patients with ATTRv amyloidosis with polyneuropathy (16.3 pg/mL vs 69.4 pg/mL, effect -53.1 pg/mL [95% confidence interval -60.5 to -45.9]). NfL levels at 18 months increased with placebo (99.5 pg/mL vs 63.2 pg/mL, effect 36.3 pg/mL [16.5-56.1]) and decreased with patisiran treatment (48.8 pg/mL vs 72.1 pg/mL, effect -23.3 pg/mL [-33.4 to -13.1]) from baseline. At 18 months, improvement in modified Neuropathy Impairment Score +7 score after patisiran treatment significantly correlated with reduced NfL (R = 0.43 [0.29-0.55]). CONCLUSIONS: Findings suggest that NfL may serve as a biomarker of nerve damage and polyneuropathy in ATTRv amyloidosis, enable earlier diagnosis of patients with ATTRv amyloidosis, and facilitate monitoring of disease progression. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that NfL levels may enable earlier diagnosis of polyneuropathy in patients with ATTRv amyloidosis and facilitate monitoring of disease progression.

Association of the transthyretin variant V122I with polyneuropathy among individuals of African ancestry.

Hereditary transthyretin-mediated (hATTR) amyloidosis is an underdiagnosed, progressively debilitating disease caused by mutations in the transthyretin (TTR) gene. V122I, a common pathogenic TTR mutation, is found in 3-4% of individuals of African ancestry in the United States and has been associated with cardiomyopathy and heart failure. To better understand the phenotypic consequences of carrying V122I, we conducted a phenome-wide association study scanning 427 ICD diagnosis codes in UK Biobank participants of African ancestry (n = 6062). Significant associations were tested for replication in the Penn Medicine Biobank (n = 5737) and the Million Veteran Program (n = 82,382). V122I was significantly associated with polyneuropathy in the UK Biobank (odds ratio [OR] = 6.4, 95% confidence interval [CI] 2.6-15.6, p = 4.2 × 10-5), which was replicated in the Penn Medicine Biobank (OR = 1.6, 95% CI 1.2-2.4, p = 6.0 × 10-3) and Million Veteran Program (OR = 1.5, 95% CI 1.2-1.8, p = 1.8 × 10-4). Polyneuropathy prevalence among V122I carriers was 2.1%, 9.0%, and 4.8% in the UK Biobank, Penn Medicine Biobank, and Million Veteran Program, respectively. The cumulative incidence of common hATTR amyloidosis manifestations (carpal tunnel syndrome, polyneuropathy, cardiomyopathy, heart failure) was significantly enriched in V122I carriers compared with non-carriers (HR = 2.8, 95% CI 1.7-4.5, p = 2.6 × 10-5) in the UK Biobank, with 37.4% of V122I carriers having at least one of these manifestations by age 75. Our findings show that V122I carriers are at increased risk of polyneuropathy. These results also emphasize the underdiagnosis of disease in V122I carriers with a significant proportion of subjects showing phenotypic changes consistent with hATTR amyloidosis. Greater understanding of the manifestations associated with V122I is critical for earlier diagnosis and treatment.

Transthyretin-stabilising mutation T119M is not associated with protection against vascular disease or death in the UK Biobank.

Background: Destabilised transthyretin (TTR) can result in the progressive, fatal disease transthyretin-mediated (ATTR) amyloidosis. A stabilising TTR mutation, T119M, is the basis for a therapeutic strategy to reduce destabilised TTR. Recently, T119M was associated with extended lifespan and lower risk of cerebrovascular disease in a Danish cohort. We aimed to determine whether this finding could be replicated in the UK Biobank.Methods: TTR T119M carriers were identified in the UK Biobank, a large prospective cohort of ∼500,000 individuals. Association between T119M genotype and inpatient diagnosis of vascular disease, cardiovascular disease, cerebrovascular disease, and mortality was analysed.Results: Frequency of T119M within the white UK Biobank population (n = 337,148) was 0.4%. Logistic regression comparing T119M carriers to non-carriers found no association between T119M and vascular disease (odds ratio [OR] = 1.08; p = .27), cardiovascular disease (OR = 1.08; p = .31), cerebrovascular disease (OR = 1.1; p = .42), or death (OR = 1.2; p = .06). Cox proportional hazards regression showed similar results (hazard ratio >1, p>.05). Age at death and vascular disease diagnosis were similar between T119M carriers and non-carriers (p = .12 and p = .38, respectively).Conclusions: There was no association between the TTR T119M genotype and risk of vascular disease or death in a large prospective cohort study, indicating that TTR tetramer stabilisation through T119M is not protective in this setting.

Characterising a healthy adult with a rare HAO1 knockout to support a therapeutic strategy for primary hyperoxaluria.

By sequencing autozygous human populations, we identified a healthy adult woman with lifelong complete knockout of HAO1 (expected ~1 in 30 million outbred people). HAO1 (glycolate oxidase) silencing is the mechanism of lumasiran, an investigational RNA interference therapeutic for primary hyperoxaluria type 1. Her plasma glycolate levels were 12 times, and urinary glycolate 6 times, the upper limit of normal observed in healthy reference individuals (n = 67). Plasma metabolomics and lipidomics (1871 biochemicals) revealed 18 markedly elevated biochemicals (>5 sd outliers versus n = 25 controls) suggesting additional HAO1 effects. Comparison with lumasiran preclinical and clinical trial data suggested she has <2% residual glycolate oxidase activity. Cell line p.Leu333SerfsTer4 expression showed markedly reduced HAO1 protein levels and cellular protein mis-localisation. In this woman, lifelong HAO1 knockout is safe and without clinical phenotype, de-risking a therapeutic approach and informing therapeutic mechanisms. Unlocking evidence from the diversity of human genetic variation can facilitate drug development.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.