Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, but not by DNA methylation, histone deacetylation, or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres accumulate DDR signals and become more accessible to telomere-associated proteins. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery.

Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules.

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 slows motility by binding to the AAA+ ring of dynein, not by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect force generation, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1-binding sites on the dynein stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1-stalk interaction slows the detachment of dynein from microtubules by interfering with the stalk sliding mechanism.

Directionality of dynein is controlled by the angle and length of its stalk.

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of dynein by engineering a Saccharomyces cerevisiae dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the dynein stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the minus end of the microtubule.

Activation and Regulation of Cytoplasmic Dynein.

Cytoplasmic dynein is an AAA+ motor that drives the transport of many intracellular cargoes towards the minus end of microtubules (MTs). Previous in vitro studies characterized isolated dynein as an exceptionally weak motor that moves slowly and diffuses on an MT. Recent studies altered this view by demonstrating that dynein remains in an autoinhibited conformation on its own, and processive motility is activated when it forms a ternary complex with dynactin and a cargo adaptor. This complex assembles more efficiently in the presence of Lis1, providing an explanation for why Lis1 is a required cofactor for most cytoplasmic dynein-driven processes in cells. This review describes how dynein motility is activated and regulated by cargo adaptors and accessory proteins.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

Cryo-EM shows how dynactin recruits two dyneins for faster movement.

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail-dynactin-BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein-dynactin transport machine.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Endothelin-converting Enzyme-1b Genetic Variants Increase the Risk of Coronary Artery Ectasia.

Coronary artery ectasia (CAE), defined as a 1.5-fold or greater enlargement of a coronary artery segment compared to the adjacent normal coronary artery, is frequently associated with atherosclerotic coronary artery disease (CAD). Membrane-bound endothelin converting enzyme-1 (ECE-1) is involved in the maturation process of the most potent vasoconstrictor ET-1. Polymorphisms in the endothelin (ET) gene family have been shown associated with the development of atherosclerosis. This study aims to investigate the effects of rs213045 and rs2038089 polymorphisms in the ECE-1 gene which have been previously shown to be associated with atherosclerosis and hypertension (HT), in CAE patients. Ninety-six CAE and 175 patients with normal coronary arteries were included in the study. ECE-1b gene variations rs213045 and rs2038089 were determined by real-time PCR. The frequencies of rs213045 C > A (C338A) CC genotype (60.4% vs. 35.4%, p < 0.001) and rs2038089 T > C T allele (64.58% vs. 35.42%, p = 0.017) were higher in the CAE group compared to the control group. The multivariate regression analysis showed that the ECE-1b rs213045 CC genotype (p = 0.001), rs2038089 T allele (p = 0.017), and hypercholesterolemia (HC) (p = 0.001) are risk factors for CAE. Moreover, in nondiabetic individuals of the CAE and control groups, it was observed that the rs213045 CC genotype (p < 0.001), and rs2038089 T allele (p = 0.003) were a risk factor for CAE, but this relationship was not found in the diabetic subgroups of the study groups (p > 0.05). These results show that ECE-1b polymorphisms may be associated with the risk of CAE and this relationship may change according to the presence of type II diabetes.

Next-generation sequencing of prolidase gene identifies novel and common variants associated with low prolidase in coronary artery ectasia.

BACKGROUND: Decreased collagen biosynthesis and increased collagenolysis can cause ectasia progression in the arterial walls. Prolidase is a key enzyme in collagen synthesis; a decrease in prolidase activity or level may decrease collagen biosynthesis, which may contribute to ectasia formation. Considering that, the variations in PEPD gene encoding prolidase enzyme were evaluated by analyzing next-generation sequencing (NGS) for the first time together with known risk factors in coronary artery ectasia (CAE) patients. METHODS: Molecular analysis of the PEPD gene was performed on genomic DNA by NGS in 76 CAE patients and 76 controls. The serum levels of prolidase were measured by the sandwich-ELISA technique. RESULTS: Serum prolidase levels were significantly lower in CAE group compared to control group, and it was significantly lower in males than females in both groups (p < 0.001). On the other hand, elevated prolidase levels were observed in CAE patients in the presence of diabetes (p < 0.001), hypertension (p < 0.05) and hyperlipidemia (p < 0.05). Logistic regression analysis demonstrated that the low prolidase level (p < 0.001), hypertension (p < 0.02) and hyperlipidemia (p < 0.012) were significantly associated with increased CAE risk. We identified four missense mutations in the PEPD gene, namely G296S, T266A, P365L and S134C (novel) that could be associated with CAE. The pathogenicity of these mutations was predicted to be "damaging" for G296S, S134C and P365L, but "benign" for T266A. We also identified a novel 5'UTR variation (Chr19:34012748 G>A) in one patient who had a low prolidase level. In addition, rs17570 and rs1061338 common variations of the PEPD gene were associated with low prolidase levels in CAE patients, while rs17569 variation was associated with high prolidase levels in both CAE and controls (p < 0.05). CONCLUSIONS: Our findings indicate that the low serum prolidase levels observed in CAE patients is significantly associated with PEPD gene variations. It was concluded that low serum prolidase level and associated PEPD mutations may be potential biomarkers for the diagnosis of CAE.

Intramolecular strain coordinates kinesin stepping behavior along microtubules.

Kinesin advances 8 nm along a microtubule per ATP hydrolyzed, but the mechanism responsible for coordinating the enzymatic cycles of kinesin's two identical motor domains remains unresolved. Here, we have tested whether such coordination is mediated by intramolecular tension generated by the "neck linkers," mechanical elements that span between the motor domains. When tension is reduced by extending the neck linkers with artificial peptides, the coupling between ATP hydrolysis and forward stepping is impaired and motor's velocity decreases as a consequence. However, speed recovers to nearly normal levels when external tension is applied by an optical trap. Remarkably, external load also induces bidirectional stepping of an immotile kinesin that lacks its mechanical element (neck linker) and fuel (ATP). Our results indicate that the kinesin motor domain senses and responds to strain in a manner that facilitates its plus-end-directed stepping and communication between its two motor domains.

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Mediating effect of work stress in the relationship between fear of COVID-19 and nurses' organizational and professional turnover intentions.

Nursing is one of the most stressful and high-risk professions. It is important to identify the psychological problems experienced by nurses during the COVID-19 pandemic and examine the relationship between these problems to devise measures that can properly address them. This study examined mediating effect of work stress in the relationship between fear of COVID-19 and nurses' organizational and professional turnover intentions. Using a cross-sectional research design, this study was conducted on 486 nurses working in seven hospitals in Turkey. The mean age of the participants was 35.24 ± 6.81 and 59.9 % of them were women. The Fear of COVID-19 Scale, the General Work Stress Scale, and the Turnover Intention Scale were used to collect data. A mediation model showed that fear of COVID-19 was positively associated with work stress and organizational and professional turnover intentions. The model also revealed that work stress was positively associated with organizational and professional turnover intentions. Furthermore, the results demonstrated that fear of COVID-19 did not only have a direct effect on organizational and professional turnover intentions but also had an indirect effect on it via increased work stress. Findings improve our understanding of the role of work stress in the relationship between fear of COVID-19 and organizational and professional turnover intentions. The findings are fruitful for tailoring and implementing intervention programs to reduce the adverse psychological impacts of COVID-19 on nurses.

SARS-CoV-2 Delta Variant Decreases Nanobody Binding and ACE2 Blocking Effectivity.

The Delta variant spreads more rapidly than previous variants of SARS-CoV-2. This variant comprises several mutations on the receptor-binding domain (RBDDelta) of its spike glycoprotein, which binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. The RBD-PD interaction has been targeted by antibodies and nanobodies to prevent viral infection, but their effectiveness against the Delta variant remains unclear. Here, we investigated RBDDelta-PD interactions in the presence and absence of nanobodies H11-H4, H11-D4, and Ty1 by performing 21.8 µs of all-atom molecular dynamics simulations. Unbiased simulations revealed that Delta variant mutations strengthen RBD binding to ACE2 by increasing the hydrophobic interactions and salt bridge formation, but weaken interactions with H11-H4, H11-D4, and Ty1. Among these nanobodies H11-H4 and H11-D4 bind RBD without overlapping ACE2. They were unable to dislocate ACE2 from RBDDelta when bound side by side with ACE2 on RBD. Steered molecular dynamics simulations at comparable loading rates to high-speed atomic force microscopy (AFM) experiments estimated lower rupture forces of the nanobodies from RBDDelta compared to ACE2. Our results suggest that existing nanobodies are less effective to inhibit RBDDelta-PD interactions and a new generation of nanobodies is needed to neutralize the Delta variant.

Cargo adaptors regulate stepping and force generation of mammalian dynein-dynactin.

Cytoplasmic dynein is an ATP-driven motor that transports intracellular cargos along microtubules. Dynein adopts an inactive conformation when not attached to a cargo, and motility is activated when dynein assembles with dynactin and a cargo adaptor. It was unclear how active dynein-dynactin complexes step along microtubules and transport cargos under tension. Using single-molecule imaging, we showed that dynein-dynactin advances by taking 8 to 32-nm steps toward the microtubule minus end with frequent sideways and backward steps. Multiple dyneins collectively bear a large amount of tension because the backward stepping rate of dynein is insensitive to load. Recruitment of two dyneins to dynactin increases the force generation and the likelihood of winning against kinesin in a tug-of-war but does not directly affect velocity. Instead, velocity is determined by cargo adaptors and tail-tail interactions between two closely packed dyneins. Our results show that cargo adaptors modulate dynein motility and force generation for a wide range of cellular functions.

Binding Mechanism of Neutralizing Nanobodies Targeting SARS-CoV-2 Spike Glycoprotein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors. Several nanobodies neutralize SARS-CoV-2 infection by binding to the receptor-binding domain (RBD) of the S protein, but how their binding antagonizes S-ACE2 interactions is not well understood. Here, we identified interactions between the RBD and nanobodies H11-H4, H11-D4, and Ty1 by performing all-atom molecular dynamics simulations. H11-H4 and H11-D4 can bind to RBD without overlapping with ACE2. H11-H4, and to a lesser extent H11-D4, binding dislocates ACE2 from its binding site due to electrostatic repulsion. In comparison, Ty1 overlaps with ACE2 on RBD and has a similar binding strength to ACE2. Mutations in the Alpha variant of SARS-CoV-2 had a minor effect in RBD binding strengths of ACE2 and nanobodies, but reduced the ability of H11-H4 and H11-D4 to dislocate ACE2 from RBD. In comparison, the Beta variant weakened the RBD binding strengths of H11-H4 and H11-D4, which were less effective to dislocate ACE2 binding. Unexpectedly, mutations in Beta strengthened Ty1 binding to RBD, suggesting that this nanobody may be more effective to neutralize the Beta variant of SARS-CoV-2.

MotAB-like machinery drives the movement of MreB filaments during bacterial gliding motility.

MreB is a bacterial actin that is important for cell shape and cell wall biosynthesis in many bacterial species. MreB also plays crucial roles in Myxococcus xanthus gliding motility, but the underlying mechanism remains unknown. Here we tracked the dynamics of single MreB particles in M. xanthus using single-particle tracking photoactivated localization microscopy. We found that a subpopulation of MreB particles moves rapidly along helical trajectories, similar to the movements of the MotAB-like gliding motors. The rapid MreB motion was stalled in the mutants that carried truncated gliding motors. Remarkably, M. xanthus MreB moves one to two orders of magnitude faster than its homologs that move along with the cell wall synthesis machinery in Bacillus subtilis and Escherichia coli, and this rapid movement was not affected by the inhibitors of cell wall biosynthesis. Our results show that in M. xanthus, MreB provides a scaffold for the gliding motors while the gliding machinery drives the movement of MreB filaments, analogous to the interdependent movements of myosin motors and actin in eukaryotic cells.

Comparison of Short and Mid-Term Mortality and Morbidity in Patients With Severe Aortic Stenosis (Intermediate/High Risk) Who Underwent Transcatheter Aortic Valve Replacement and Surgical Aortic Valve Replacement.

Treatment protocols for severe aortic valve stenosis include surgical aortic valve replacement (SAVR), balloon valvuloplasty, transcatheter aortic valve replacement (TAVR), and medical  treatment. Because the success rates are getting higher with both SAVR and TAVR, making the right treatment decision is important. This study retrospectively shows the short- (1 month) and mid-term (6 months) mortality and morbidity rate differences between 2 groups of patients, who arrived to our hospital from January 2014 through October 2018. The first group consists of 54 patients who underwent mid-high risk SAVR operations at Istanbul University-Cerrahpasa, Institute of Cardiology, Department of Cardiovascular Surgery. The second group consists of 57 patients who underwent TAVR at the Cardiology Department. Preoperative evaluation showed that the mean age of the SAVR group (71.5 years) was higher than the TAVR group (80 years). Also, the history of previous cardiac valve replacement surgery significantly was higher in the SAVR group than the TAVR group (P = .028). There were no significant differences between the remaining preoperative tests and diagnostic procedures. Of the patients who underwent SAVR, 3.7% experienced postoperative cardiac arrhythmias, while the 17.5% of patients from the TAVR group experienced cardiac arrhythmias after the procedure. This difference between the groups were statistically significant. Mortality rate was 9.3% in the SAVR group and 5.3% in the TAVR group. The mortality rate was not statistically different between the groups. There was no significant difference between the groups in the means of neurological incidents. The TAVR group had more vascular complications (17.9% to none) and pacemaker implantations (21.4% to 1.9%). Minor or major bleeding was the most common reason for admission to the hospital after SAVR. Seven out of 10 patients experienced bleeding. Aortic regurgitation was more common in the TAVR group at the first and sixth month following the procedure. Ratios between the gradient values were higher in the SAVR group (P < .001). Peak gradient values at the sixth month following the procedure were lower than the values of the first month (P < .040). Aortic regurgitation symptoms increased with patients at the mid-term follow-up appointment. To prevent the vascular complications in the TAVR group, preoperative peripheral vascular examination thoroughly should be performed. Considering that bleeding disorders are the main reason the SAVR group arrived to the hospital, INR values should closely be monitored. There seems to be no mortality difference between the groups at the six-month follow up, but studies should continue with more patients and long-term results.