Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

Latest developments in early diagnosis and specific treatment of severe influenza infection.

Influenza pandemics are unpredictable recurrent events with global health, economic, and social consequences. The objective of this review is to provide an update on the latest developments in early diagnosis and specific treatment of the disease and its complications, particularly with regard to respiratory organ failure. Despite advances in treatment, the rate of mortality in the intensive care unit remains approximately 30%. Therefore, early identification of potentially severe viral pneumonia is extremely important to optimize treatment in these patients. The pathogenesis of influenza virus infection depends on viral virulence and host response. Thus, in some patients, it is associated with an excessive systemic response mediated by an authentic cytokine storm. This process leads to severe primary pneumonia and acute respiratory distress syndrome. Initial prognostication in the emergency department based on comorbidities, vital signs, and biomarkers (e.g., procalcitonin, ferritin, human leukocyte antigen-DR, mid-regional proadrenomedullin, and lactate) is important. Identification of these biomarkers on admission may facilitate clinical decision-making to determine early admission to the hospital or the intensive care unit. These decisions are reached considering pathophysiological circumstances that are associated with a poor prognosis (e.g., bacterial co-infection, hyperinflammation, immune paralysis, severe endothelial damage, organ dysfunction, and septic shock). Moreover, early implementation is important to increase treatment efficacy. Based on a limited level of evidence, all current guidelines recommend using oseltamivir in this setting. The possibility of drug resistance should also be considered. Alternative options include other antiviral drugs and combination therapies with monoclonal antibodies. Importantly, it is not recommended to use corticosteroids in the initial treatment of these patients. Furthermore, the implementation of supportive measures for respiratory failure is essential. Current recommendations are limited, heterogeneous, and not regularly updated. Early intubation and mechanical ventilation is the basic treatment for patients with severe respiratory failure. Prone ventilation should be promptly performed in patients with acute respiratory distress syndrome, while early tracheostomy should be considered in case of planned prolonged mechanical ventilation. Clinical trials on antiviral treatment and respiratory support measures specifically for these patients, as well as specific recommendations for different at-risk populations, are necessary to improve outcomes.

VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates.

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

Substrate recognition principles for the PP2A-B55 protein phosphatase.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that α-helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α-helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting complex by binding to an α-helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease.

Does a simplified algorithm and integrated HCV care model improve linkage to care, retention, and cure among people who inject drugs? A pragmatic quality improvement randomized controlled trial protocol.

BACKGROUND: As many as 2.4 million Americans are affected by chronic Hepatitis C Virus (HCV) in the United States.In 2018, the estimated number of adults with a history of HCV infection in San Diego County was 55,354 (95% CI: 25,411-93,329). This corresponded to a seroprevalence of 2.1% (95% CI: 2.1-3.4%). One-third of infections were among PWID. Published research has demonstrated that direct-acting antivirals (DAAs) have high efficacy and can now be used by primary care providers to treat HCV. In addition, limited evidence exists to support the effectiveness of simplified algorithms in clinical trial and real-world settings. Even with expanded access to HCV treatment in primary care settings, there are still groups, especially people who inject drugs (PWID) and people experiencing homelessness, who experience treatment disparities due to access and treatment barriers. The current study extends the simplified algorithm with a streetside 'one-stop-shop' approach with integrated care (including the offer of buprenorphine prescriptions and abscess care) using a mobile clinic situated adjacent to a syringe service program serving many homeless populations. Rates of HCV treatment initiation and retention will be compared between patients offered HCV care in a mobile clinic adjacent to a syringe services program (SSP) and homeless encampment versus those who are linked to a community clinic's current practice of usual care, which includes comprehensive patient navigation. METHODS: A quasi-experimental, prospective, interventional, comparative effectiveness trial with allocation of approximately 200 patients who inject drugs and have chronic HCV to the "simplified care" pathway (intervention group) or the "usual care" pathway (control group). Block randomization will be performed with a 1:1 randomization. DISCUSSION: Previous research has demonstrated acceptable outcomes for patients treated using simplified algorithms for DAAs and point-of-care testing in mobile medical clinics; however, there are opportunities to explore how these new, innovative systems of care impact treatment initiation rates or other HCV care cascade outcomes among PWID. TRIAL REGISTRATION: We have registered our study with ClinicalTrials.gov, a resource of the United States National Library of Medicine. This database contains research studies from United States and other countries around the world. Our study has not been previously published. The ClinicalTrials.gov registration identifier is NCT04741750.

The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.

Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification.

Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A-B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A-B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation.

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

Spatial separation of phosphatase and kinase activity within the Bub complex is required for proper mitosis.

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

Molecular basis of cyclic tetra-oligoadenylate processing by small standalone CRISPR-Cas ring nucleases.

Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition.

Host response dysregulations amongst adults hospitalized by influenza A H1N1 virus pneumonia: A prospective multicenter cohort study.

BACKGROUND: Limited knowledge exists on how early host response impacts outcomes in influenza pneumonia. METHODS: This study assessed what was the contribution of host immune response at the emergency department on hospital mortality amongst adults with influenza A H1N1pdm09 pneumonia and whether early stratification by immune host response anticipates the risk of death. This is a secondary analysis from a prospective, observational, multicenter cohort comparing 75 adults requiring intensive care with 38 hospitalized in medical wards. Different immune response biomarkers within 24 h of hospitalization and their association with hospital mortality were assessed. RESULTS: Fifty-three were discharged alive. Non-survivors were associated (p<0.05) with lower lymphocytes (751 vs. 387), monocytes (450 vs. 220) expression of HLA-DR (1,662 vs. 962) and higher IgM levels (178 vs. 152;p<0.01). Lymphocyte subpopulations amongst non-survivors showed a significantly (p<0.05) lower number of TCD3+ (247.2 vs. 520.8), TCD4+ (150.3 vs. 323.6), TCD8+ (95.3 vs. 151.4) and NKCD56+ (21.9 vs. 91.4). Number of lymphocytes, monocytes and NKCD56+ predicted hospital mortality (AUC 0.854). Hospital mortality was independently associated with low HLA-DR values, low number of NKCD56+ cells, and high IgM levels, in a Cox-proportional hazard analysis. A second model, documented that hospital mortality was independently associated with a phenotype combining immunoparalysis with hyperinflammation (HR 5.53; 95%CI 2.16-14.14), after adjusting by predicted mortality. CONCLUSIONS: We conclude that amongst influenza pneumonia, presence of immunoparalysis was a major mortality driver. Influenza heterogeneity was partly explained by early specific host response dysregulations which should be considered to design personalized approaches of adjunctive therapy.

Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening.

CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.

Plasma Levels of Mid-Regional Proadrenomedullin Accurately Identify H1N1pdm09 Influenza Virus Patients with Risk of Intensive Care Admission and Mortality in the Emergency Department.

Early identification of severe viral pneumonia in influenza virus A (H1N1pdm09) patients is extremely important for prompt admission to the ICU. The objective is to evaluate the usefulness of MR-proadrenomedullin (MR-proADM) compared to C reactive protein (CRP), procalcitonin (PCT), and ferritin in the prognosis of influenza A pneumonia. This prospective, observational, multicenter study included one hundred thirteen patients with confirmed influenza virus A (H1N1pdm09) admitted to an Emergency Department and ICUs of six hospitals in Spain. Measurements and Main Results: one-hundred thirteen patients with confirmed influenza virus A (H1N1pdm09) were enrolled. Seventy-five subjects (mortality 29.3%) with severe pneumonia caused by influenza A H1N1pdm09 virus (H1N1vIPN) were compared with 38 controls (CG).The median MR-proADM levels at hospital admission were 1.2 nmol/L (IQR (0.8-2.6) vs. 0.5 nmol/L (IQR 0.2-0.9) in the CG (p = 0.01), and PCT levels were 0.43 µg/L (IQR 0.2-1.2) in the H1N1vIPN group and 0.1 µg/L (IQR 0.1-0.2) in the CG (p < 0.01). CRP levels at admission were 15.5 mg/dL(IQR 9.2-24.9) in H1N1vIPN and 8.6 mg/dL(IQR 3-17.3) in the CG (p < 0.01). Ferritin levels at admission were 558.1 ng/mL(IQR 180-1880) in H1N1vIPN and 167.7 ng/mL(IQR 34.8-292.9) in the CG (p < 0.01). A breakpoint for hospital admission of MR-proADM of 1.1 nmol/L showed a sensitivity of 55% and a specificity of 90% (AUC-ROC0.822). Non-survivors showed higher MR-proADM levels: median of 2.5 nmol/L vs. 0.9 nmol/L among survivors (p < 0.01). PCT, CRP, and ferritin levels also showed significant differences in predicting mortality. The MR-proADM AUC-ROC for mortality was 0.853 (p < 0.01). In a Cox proportional hazards model, MR-proADM levels > 1.2 nmol/L at hospital admission were significant predictive factors for ICU and 90-day mortality (HR: 1.3). Conclusions: the initial MR-proADM, ferritin, CRP, and PCT levels effectively determine adverse outcomes and risk of ICU admission and mortality in patients with influenza virus pneumonia. MR-proADM has the highest potency for survival prediction.

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination.

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

Microscale Thermophoresis and additional effects measured in NanoTemper Monolith instruments.

NanoTemper Monolith instruments have gained enormous popularity for measuring molecular interactions both in academia and industry. The underlying technology has been extensively reviewed along with its assumptions, limitations, and applications (Scheuermann et al., Anal Biochem 496:79-93, 2016). Several assumptions about the technique such as the extent of thermal deviations generated by the infrared laser and the thermophoretic foundation of the measured signal have been revised during the last decade. We present here in this letter the experience gathered in scientific service facilities about this technique and make scientists aware of possible pitfalls with the intention to promote knowledge and good practice throughout the scientific community.