Identification of an Optimized Receptor-Binding Domain Subunit Vaccine against SARS-CoV-2.

Current vaccine efforts to combat SARS-CoV-2 are focused on the whole spike protein administered as mRNA, viral vector, or protein subunit. However, the SARS-CoV-2 receptor-binding domain (RBD) is the immunodominant portion of the spike protein, accounting for 90% of serum neutralizing activity. In this study, we constructed several versions of RBD and together with aluminum hydroxide or DDA (dimethyldioctadecylammonium bromide)/TDB (d-(+)-trehalose 6,6'-dibehenate) adjuvant evaluated immunogenicity in mice. We generated human angiotensin-converting enzyme 2 knock-in mice to evaluate vaccine efficacy in vivo following viral challenge. We found that 1) subdomain (SD)1 was essential for the RBD to elicit maximal immunogenicity; 2) RBDSD1 produced in mammalian HEK cells elicited better immunogenicity than did protein produced in insect or yeast cells; 3) RBDSD1 combined with the CD4 Th1 adjuvant DDA/TDB produced higher neutralizing Ab responses and stronger CD4 T cell responses than did aluminum hydroxide; 4) addition of monomeric human Fc receptor to RBDSD1 (RBDSD1Fc) significantly enhanced immunogenicity and neutralizing Ab titers; 5) the Beta version of RBDSD1Fc provided a broad range of cross-neutralization to multiple antigenic variants of concern, including Omicron; and 6) the Beta version of RBDSD1Fc with DDA/TDB provided complete protection against virus challenge in the knock-in mouse model. Thus, we have identified an optimized RBD-based subunit vaccine suitable for clinical trials.

Oxygen-dependent Regulation of Erythropoietin Receptor Turnover and Signaling.

von Hippel-Lindau (VHL) disease is a rare familial cancer predisposition syndrome caused by a loss or mutation in a single gene,VHL, but it exhibits a wide phenotypic variability that can be categorized into distinct subtypes. The phenotypic variability has been largely argued to be attributable to the extent of deregulation of the α subunit of hypoxia-inducible factor α, a well established target of VHL E3 ubiquitin ligase, ECV (Elongins/Cul2/VHL). Here, we show that erythropoietin receptor (EPOR) is hydroxylated on proline 419 and 426 via prolyl hydroxylase 3. EPOR hydroxylation is required for binding to the ß domain of VHL and polyubiquitylation via ECV, leading to increased EPOR turnover. In addition, several type-specific VHL disease-causing mutants, including those that have retained proper binding and regulation of hypoxia-inducible factor α, showed a severe defect in binding prolyl hydroxylated EPOR peptides. These results identify EPOR as the secondbona fidehydroxylation-dependent substrate of VHL that potentially influences oxygen homeostasis and contributes to the complex genotype-phenotype correlation in VHL disease.

A multi-specific, multi-affinity antibody platform neutralizes sarbecoviruses and confers protection against SARS-CoV-2 in vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

One swab, two tests: Validation of dual SARS-CoV-2 testing on the Abbott ID NOW™.

BACKGROUND: Point-of-care tests (POCT) are promising tools to detect SARS-CoV-2 in specific settings. Initial reports suggest the ID NOW™ COVID-19 assay (Abbott Diagnostics Inc, USA) is less sensitive than standard real-time reverse transcription polymerase chain reaction (rRT-PCR) assays. This has raised concern over false negatives in SARS-CoV-2 POCT. OBJECTIVES: We compared the performance of the ID NOW™ COVID-19 assay to our in-house rRT-PCR assay to assess whether dry swabs used in ID NOW™ testing could be stored in transport media and be re-tested by rRT-PCR for redundancy and to provide material for further investigation. METHODS: Paired respiratory swabs collected from patients at three acute care hospitals were used. One swab in transport media (McMaster Molecular Media (MMM)) was tested for SARS-CoV-2 by a laboratory-developed two-target rRT-PCR assay. The second was stored dry in a sterile container and tested by the ID NOW™ COVID-19 assay. Following ID NOW™ testing, dry swabs were stored in MMM for up to 48 h and re-tested by rRT-PCR. Serially diluted SARS-CoV-2 particles were used to assess the impact of heat inactivation and storage time. RESULTS: Respiratory swabs (n = 343) from 179 individuals were included. Using rRT-PCR results as the comparator, the ID NOW™ COVID-19 assay had positive (PPA) and negative (NPA) percent agreements of 87.0% (95% CI:0.74-0.94) and 99.7% (95% CI:0.98-0.99). Re-tested swabs placed in MMM following ID NOW testing had PPA and NPA of 88.8% (95% CI:0.76-0.95) and 99.7% (95% CI:0.98-0.99), respectively. CONCLUSIONS: Storing spent dry swabs in transport media for redundancy rRT-PCR testing is a potential approach to address possible false negatives with the ID NOW™ COVID-19 assay.

Vapourized hydrogen peroxide decontamination in a hospital setting inactivates SARS-CoV-2 and HCoV-229E without compromising filtration efficiency of unexpired N95 respirators.

BACKGROUND: The COVID-19 pandemic highlighted the need for evidence-based approaches to decontamination and reuse of N95 filtering facepiece respirators (FFRs). We sought to determine whether vapourized hydrogen peroxide (VHP) reduced SARS-CoV-2 bioburden on FFRs without compromising filtration efficiency. We also investigated coronavirus HCoV-229E as a surrogate for decontamination validation testing. METHODS: N95 FFRs were laced with SARS-CoV-2 or HCoV-229E and treated with VHP in a hospital reprocessing facility. After sterilization, viral burden was determined using viral outgrowth in a titration assay, and filtration efficiency of FFRs was tested against ATSM F2299 and NIOSH TEB-STP-APR-0059. RESULTS: Viable SARS-CoV-2 virus was not detected after VHP treatment. One replicate of the HCoV-229E laced FFRs yielded virus after processing. Unexpired N95 FFRs retained full filtration efficiency after VHP processing. Expired FFRs failed to meet design-specified filtration efficiency and therefore are unsuitable for reprocessing. DISCUSSION: In-hospital VHP is an effective decontaminant for SARS-CoV-2 on FFRs. Further, filtration efficiency of unexpired respirators is not affected by this decontamination process. CONCLUSIONS: VHP is effective in inactivating SARS-CoV-2 on FFRs without compromising filtration efficiency. HCoV-229E is a suitable surrogate for SARS-CoV-2 for disinfection studies.

The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors.

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

Intranasal HD-Ad vaccine protects the upper and lower respiratory tracts of hACE2 mice against SARS-CoV-2.

BACKGROUND: The ongoing COVID-19 pandemic has resulted in 185 million recorded cases and over 4 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. RESULTS: Here, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) produces a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. CONCLUSION: Our approaches provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

Tripartite purinergic modulation of central respiratory networks during perinatal development: the influence of ATP, ectonucleotidases, and ATP metabolites.

ATP released during hypoxia from the ventrolateral medulla activates purinergic receptors (P2Rs) to attenuate the secondary hypoxic depression of breathing by a mechanism that likely involves a P2Y(1)R-mediated excitation of preBötzinger complex (preBötC) inspiratory rhythm-generating networks. In this study, we used rhythmically active in vitro preparations from embryonic and postnatal rats and ATP microinjection into the rostral ventral respiratory group (rVRG)/preBötC to reveal that these networks are sensitive to ATP when rhythm emerges at embryonic day 17 (E17). The peak frequency elicited by ATP at E19 and postnatally was the same ( approximately 45 bursts/min), but relative sensitivity was threefold greater at E19, reflecting a lower baseline frequency (5.6 +/- 0.9 vs 19.0 +/- 1.3 bursts/min). Combining microinjection techniques with ATP biosensors revealed that ATP concentration in the rVRG/preBötC falls rapidly as a result of active processes and closely correlates with inspiratory frequency. A phosphate assay established that preBötC-containing tissue punches degrade ATP at rates that increase perinatally. Thus, the agonist profile [ATP/ADP/adenosine (ADO)] produced after ATP release in the rVRG/preBötC will change perinatally. Electrophysiology further established that the ATP metabolite ADP is excitatory and that, in fetal but not postnatal animals, ADO at A(1) receptors exerts a tonic depressive action on rhythm, whereas A(1) antagonists extend the excitatory action of ATP on inspiratory rhythm. These data demonstrate that ATP is a potent excitatory modulator of the rVRG/preBötC inspiratory network from the time it becomes active and that ATP actions are determined by a dynamic interaction between the actions of ATP at P2 receptors, ectonucleotidases that degrade ATP, and ATP metabolites on P2Y and P1 receptors.

Generation of eupnea and sighs by a spatiochemically organized inspiratory network.

The discovery of the rhythmogenic pre-Bötzinger complex (preBötC) inspiratory network, which remains active in a transverse brainstem slice, greatly increased the understanding of neural respiratory control. However, basic questions remain unanswered such as (1) What are the necessary and sufficient slice boundaries for a functional preBötC? (2) Is the minimal preBötC capable of reconfiguring between inspiratory-related patterns (e.g., fictive eupnea and sighs)? (3) How is preBötC activity affected by surrounding structures? Using newborn rat slices with systematically varied dimensions in physiological [K(+)] (3 mM), we found that a 175 microm thickness is sufficient for generating inspiratory-related rhythms. In 700-microm-thick slices with unilaterally exposed preBötC, a kernel <100 microm thick, centered 0.5 mm caudal to the facial nucleus, is necessary for rhythm generation. Slices containing this kernel plus caudal structures produced eupneic bursts of regular amplitude, whereas this kernel plus rostral tissue generated sighs, intermingled with eupneic bursts of variable amplitude ("eupnea-sigh pattern"). After spontaneous arrest of rhythm, substance-P or neurokinin-1 (NK1) receptor agonist induced the eupnea-sigh burst pattern in > or = 250-microm-thick slices, whereas thyrotropin-releasing hormone or phosphodiesterase-4 blockers evoked the eupnea burst pattern. Endogenous rhythm was depressed by NK1 receptor antagonism. Multineuronal Ca(2+) imaging revealed that preBötC neurons reconfigure between eupnea and eupnea-sigh burst patterns. We hypothesize a (gradient-like) spatiochemical organization of regions adjacent to the preBötC, such that a small preBötC inspiratory-related oscillator generates eupnea under the dominant influence of caudal structures or thyrotropin-releasing hormone-like transmitters but eupnea-sigh activity when the influence of rostral structures or substance-P-like transmitters predominates.

A Hypoxia-Inducible HIF1-GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell-Platelet Binding.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide-negative counterparts, which protects ccRCC cells against natural killer cell-mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. IMPLICATIONS: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation.

Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled 'dark state'.

Consequences of VHL Loss on Global DNA Methylome.

In clear-cell renal cell carcinoma (ccRCC), loss of von Hippel-Lindau (VHL) tumour suppressor gene and reduced oxygen tension promote stabilisation of hypoxia-inducible factor (HIF) family of transcription factors, which promote changes in the expression of genes that contribute to oncogenesis. Multiple studies have demonstrated significant perturbations in DNA methylation in ccRCC via largely unclear mechanisms that modify the transcriptional output of tumour cells. Here, we show that the methylation status of the CpG dinucleotide within the consensus hypoxia-responsive element (HRE) markedly influences the binding of HIF and that the loss of VHL results in significant alterations in the DNA methylome. Surprisingly, hypoxia, which likewise promotes HIF stabilisation and activation, has relatively few effects on global DNA methylation. Gene expression analysis of ccRCC patient samples highlighted expression of a group of genes whose transcription correlated with methylation changes, including hypoxic responsive genes such as VEGF and TGF. These results suggest that the loss of VHL alters DNA methylation profile across the genome, commonly associated with and contributing to ccRCC progression.

High sensitivity to neuromodulator-activated signaling pathways at physiological [K+] of confocally imaged respiratory center neurons in on-line-calibrated newborn rat brainstem slices.

The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8-10 mM) superfusate [K+], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel "on-line histology" based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0-4. At physiological superfusate [K+] (3 mM), 500- and 600-microm-thick slices with the PBC in the center and the caudal boundary 0.70 and 0.76 mm caudal to the facial motonucleus generated rhythm for >2 and approximately 4 h, respectively. Rhythm was abolished by low nanomolar concentrations of the mu-opiate receptor agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin). After spontaneous arrest of bursting, rhythm was reactivated at clinically relevant or physiological concentrations by 3,5-dihydroxyphenylglycine, thyrotropin-releasing hormone, or rolipram, each affecting distinct second-messenger pathways. Two-photon/confocal Ca2+ imaging revealed that these agents reactivated the same PBC neurons initially active in 3 mM [K+]. The data show that "calibrated" PBC slices at physiological [K+] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous "in vitro apnea" is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure-function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K+].

Highlights from the Third Biennial Strategies for an HIV Cure Meeting: 14-16 November 2016, Bethesda, MD, USA.

Since the first Strategies for an HIV Cure Meeting organised by the National Institute of Allergy and Infectious Diseases (NIAID) in 2012, one of the primary purposes of the meeting has been to facilitate communication and foster collaboration across the NIAID-funded Martin Delaney Collaboratories for HIV cure research (MDC), the broader HIV cure-related research field, and industry and community stakeholders. This year's meeting agenda reflected NIAID's increasing investment over the last 5 years in research to identify strategies for eradicating or achieving long-term remission of HIV infection. Overviews and research highlights were presented from each of the Martin Delaney Collaboratories, as well as projects funded through the Beyond HAART programme, the Consortia for Innovative AIDS Research in Nonhuman Primates (CIAR) programme, the ACTG and IMPAACT clinical trial networks, and the NIAID Vaccine Research Center in hopes of stimulating cross-talk and synergy among these and other programmes focused on HIV cure research. Aside from the oral presentations described here, the meeting also included 75 poster presentations. Finally, community engagement activities and community participation in the MDC was highlighted throughout the first day and in a special session on Day 2. This reflects NIAID's commitment to engage community partners in the earliest stages of research towards curative interventions through the MDC programme. The entire meeting is available for viewing via the NIH VideoCast website at: https://videocast.nih.gov/PastEvents.asp.

Complexity of inducible nitric oxide synthase: cellular source determines benefit versus toxicity.

BACKGROUND: Inducible nitric oxide synthase (iNOS) has been shown to have both beneficial and detrimental effects in sepsis. We focused on a single organ, the heart, and used 2 distinct cell types that express iNOS-the cardiac myocyte and the infiltrating neutrophil-to study the distinct functional effects of iNOS derived from heterogeneous cellular sources. METHODS AND RESULTS: In the first series of experiments, extravascular neutrophils were exposed to isolated single endotoxemic cardiac myocytes. Adhesion of wild-type neutrophils caused a rapid decrease in myocyte shortening and a concomitant increase in neutrophil-derived intracellular oxidative stress within the myocytes that was not observed with neutrophils from iNOS-deficient animals. We previously demonstrated that neutrophil-derived superoxide was essential for myocyte dysfunction; however, superoxide production was not compromised in the iNOS-deficient neutrophils. Because both superoxide and NO were essential for the neutrophil dysfunction, we probed for but could not detect any peroxynitrite assessed by detection of nitrotyrosine. There was a significant increase in length shortening in response to beta-adrenergic stimulation of wild-type myocytes. Surprisingly, myocyte iNOS activity was essential rather than detrimental for the development of beta-adrenergic receptor-mediated increases in shortening in endotoxemic iNOS-deficient myocytes. CONCLUSIONS: These results demonstrate that iNOS, when expressed in isolated cardiac myocytes, can regulate the response to beta-adrenergic stimulation during sepsis. However, as the neutrophils migrate in proximity to myocytes, iNOS now becomes essential for the ability of neutrophils to damage myocytes. These findings demonstrate that cellular source strongly modulates the beneficial and detrimental effect of iNOS.

Rapid size dependent deletion of foreign gene sequences inserted into attenuated HIV-1 upon infection in vivo: implications for vaccine development.

Live attenuated HIV vaccines offer a means to introduce exogenous sequences into the viral genome to target the virus elimination in vivo. Foreign genes inserted into the nef region of HIV-1 NL4-3 were found to be rapidly deleted following virus infection and/or replication, in a size dependent manner, in the human fetal Thymus/Liver implants of severe combined immunodeficient mouse (SCID-hu) model. When the murine heat stable antigen (HSA) of 283 bp was substituted into HIV-1 nef region, the viral loads in vivo were comparable to the negative control nef attenuated HIV-1, and the reporter HSA gene was not deleted upon infection. However, the murine Thy1.2 gene (505 bp) substituted into the nef attenuated HIV-1, upon infection and replication, deleted 441 bp in vitro and 437 bp in vivo, of the inserted Thy1.2 gene. When the enhanced green fluorescence protein (eGFP) gene (720 bp) was substituted for nef, virus replication was aborted in vivo in the Thy/Liv implants, as seen by the background levels of viral loads, comparable to mock infected implants, and the eGFP gene was deleted. When the herpes simplex virus thymidine kinase gene, HSV-TK (1.15 kbp), or HSA gene, was substituted into the viral vpr gene, TK but not HSA gene was deleted, upon infection in vitro. Moreover, NL-TKI reporter virus with both intact nef and vpr genes shows deletion of TK gene both in vitro and in vivo. Excision of foreign genes occurred within the exogenous segments but not in the viral own regions. These results suggest that larger "suicide" genes introduced via HIV-1 can be deleted upon infection. However, smaller size nucleotide sequences or genes (approximately 300 bp) inserted in place of viral nef or vpr gene may be used to target the virus or its components, for attack and elimination in vivo, and thus have implications for the development of live attenuated HIV vaccines.

Roles for Pbp1 and caloric restriction in genome and lifespan maintenance via suppression of RNA-DNA hybrids.

Intergenic transcription within repetitive loci such as the ribosomal DNA (rDNA) repeats of yeast commonly triggers aberrant recombination. Major mechanisms suppressing aberrant rDNA recombination rely on chromatin silencing or RNAPII repression at intergenic spacers within the repeats. We find ancient processes operating at rDNA intergenic spacers and other loci to maintain genome stability via repression of RNA-DNA hybrids. The yeast Ataxin-2 protein Pbp1 binds noncoding RNA, suppresses RNA-DNA hybrids, and prevents aberrant rDNA recombination. Repression of RNA-DNA hybrids in Pbp1-deficient cells through RNaseH overexpression, deletion of the G4DNA-stabilizing Stm1, or caloric restriction operating via RNaseH/Pif1 restores rDNA stability. Pbp1 also limits hybrids at non-rDNA G4DNA loci including telomeres. Moreover, cells lacking Pbp1 have a short replicative lifespan that is extended upon hybrid suppression. Thus, we find roles for Pbp1 in genome maintenance and reveal that caloric restriction counteracts Pbp1 deficiencies by engaging RNaseH and Pif1.

DCNL1 functions as a substrate sensor and activator of cullin 2-RING ligase.

Substrate engagement by F-box proteins promotes NEDD8 modification of cullins, which is necessary for the activation of cullin-RING E3 ubiquitin ligases (CRLs). However, the mechanism by which substrate recruitment triggers cullin neddylation remains unclear. Here, we identify DCNL1 (defective in cullin neddylation 1-like 1) as a component of CRL2 called ECV (elongins BC/CUL2/VHL) and show that molecular suppression of DCNL1 attenuates CUL2 neddylation. DCNL1 via its DAD patch binds to CUL2 but is also able to bind VHL independent of CUL2 and the DAD patch. The engagement of the substrate hypoxia-inducible factor 1α (HIF1α) to the substrate receptor VHL increases DCNL1 binding to VHL as well as to CUL2. Notably, an engineered mutant form of HIF1α that associates with CUL2, but not DCNL1, fails to trigger CUL2 neddylation and retains ECV in an inactive state. These findings support a model in which substrate engagement prompts DCNL1 recruitment that facilitates the initiation of CUL2 neddylation and define DCNL1 as a "substrate sensor switch" for ECV activation.

Effects of Perinuclear Chromosome Tethers in the Telomeric URA3/5FOA System Reflect Changes to Gene Silencing and not Nucleotide Metabolism.

Telomeres are repetitive DNA sequences that protect the ends of linear chromosomes. Telomeres also recruit histone deacetylase complexes that can then spread along chromosome arms and repress the expression of subtelomeric genes in a process known as telomere position effect (TPE). In the budding yeast Saccharomyces cerevisiae, association of telomeres with the nuclear envelope is thought to promote TPE by increasing the local concentration of histone deacetylase complexes at chromosome ends. Importantly, our understanding of TPE stems primarily from studies that employed marker genes inserted within yeast subtelomeres. In particular, the prototrophic marker URA3 is commonly used to assay TPE by negative selection on media supplemented with 5-fluoro-orotic acid (5FOA). Recent findings suggested that decreased growth on 5FOA-containing media may not always indicate increased expression of a telomeric URA3 reporter, but can rather reflect an increase in ribonucleotide reductase (RNR) function and nucleotide metabolism. Thus, we set out to test if the 5FOA sensitivity of subtelomeric URA3-harboring cells in which we deleted various factors implicated in perinuclear telomere tethering reflects changes to TPE and/or RNR. We report that RNR inhibition restores 5FOA resistance to cells lacking RNR regulatory factors but not any of the major telomere tethering and silencing factors, including Sir2, cohibin, Mps3, Heh1, and Esc1. In addition, we find that the disruption of tethering pathways in which these factors participate increases the level of URA3 transcripts originating from the telomeric reporter gene and abrogates silencing of subtelomeric HIS3 reporter genes without altering RNR gene expression. Thus, increased 5FOA sensitivity of telomeric URA3-harboring cells deficient in telomere tethers reflects the dysregulation of TPE but not RNR. This is key to understanding relationships between telomere positioning, chromatin silencing, and lifespan.

Cohesin and related coiled-coil domain-containing complexes physically and functionally connect the dots across the genome.

Interactions between genetic regions located across the genome maintain its three-dimensional organization and function. Recent studies point to key roles for a set of coiled-coil domain-containing complexes (cohibin, cohesin, condensin and monopolin) and related factors in the regulation of DNA-DNA connections across the genome. These connections are critical to replication, recombination, gene expression as well as chromosome segregation.