POT1-TPP1 binding stabilizes POT1, promoting efficient telomere maintenance.

Telomeric POT1-TPP1 binding is critical to telomere maintenance and disruption of this complex may lead to cancer. Current data suggests a reduction of intracellular POT1 levels in the absence of TPP1. Here we provide evidence of POT1 plasticity that contributes to its lack of stability in the absence of TPP1 binding. Structural data reveals inter- and intramolecular POT1C domain flexibility in the absence of TPP1. Thermostability and proteolytic resistance assays show that POT1C and the mutant complex POT1C(Q623H)-TPP1(PBD) are less stable than the wild type POT1C-TPP1(PBD), suggesting that TPP1 binding to POT1 stabilizes POT1C and makes it less accessible to proteasomal degradation in the cell. Disruption of the POT1-TPP1 complex such as through cancer-associated mutations leads to a reduction of intracellular POT1, telomere uncapping, and telomere associated DNA damage response (DDR). DDR in turn leads to senescence or genomic instability and oncogenesis.

Quantification of natural abundance NMR data differentiates the solution behavior of monoclonal antibodies and their fragments.

Biotherapeutics are an important class of molecules for the treatment of a wide range of diseases. They include low molecular weight peptides, highly engineered protein scaffolds and monoclonal antibodies. During their discovery and development, assessments of the biophysical attributes is critical to understanding the solution behavior of therapeutic proteins and for de-risking liabilities. Thus, methods that can quantify, characterize, and provide a basis to inform risks and drive the selection of more optimal antibody and alternative scaffolds are needed. Nuclear Magnetic Resonance (NMR) spectroscopy is a technique that provides a means to probe antibody and antibody-like molecules in solution, at atomic resolution, under any formulated conditions. Here, all samples were profiled at natural abundance requiring no isotope enrichment. We present a numerical approach that quantitates two-dimensional methyl spectra. The approach was tested with a reference dataset that contained different types of antibody and antibody-like molecules. This dataset was processed through a procedure we call a Random Sampling of NMR Peaks for Covariance Analysis. This analysis revealed that the first two components were well correlated with the hydrodynamic radius of the molecules included in the reference set. Higher-order principal components were also linked to dynamic features between different tethered antibody-like molecules and contributed to decisions around candidate selection. The reference set provides a basis to characterize molecules with unknown solution behavior and is sensitive to the behavior of a molecule formulated under different conditions. The approach is independent of protein design, scaffold, formulation and provides a facile method to quantify solution behavior.

Cerebrovascular Complication and Valve Surgery in Infective Endocarditis.

Infective endocarditis (IE) with neurologic complications is common in patients with active IE. The most common and feared neurological complication of left-sided IE is cerebrovascular, from septic emboli causing ischemic stroke, intracranial hemorrhage (ICH), or an infectious intracranial aneurysm with or without rupture. In patients with cerebrovascular complications, valve replacement surgery is often delayed for concern of further neurological worsening. However, in circumstances when an indication for valve surgery to treat IE is present, the benefits of early surgical treatment may outweigh the potential neurologic deterioration. Furthermore, valve surgery has been associated with lower in-hospital mortality than medical therapy with intravenous antibiotics alone. Early valve surgery can be performed within 7 days of transient ischemic attack or asymptomatic stroke when medically indicated. Timing of valve surgery for IE after symptomatic medium or large symptomatic ischemic stroke or ICH remains challenging, and current data in the literature are conflicting about the risks and benefits. A delay of 2 to 4 weeks from the time of the cerebrovascular event is often recommended, balancing the risks and benefits of surgery. The range of timing of valve surgery varies depending on the clinical scenario, and is best determined by a multidisciplinary decision between cardiothoracic surgeons, cardiologists, infectious disease experts, and vascular neurologists in an experienced referral center.

Brain Injury Is More Common in Venoarterial Extracorporeal Membrane Oxygenation Than Venovenous Extracorporeal Membrane Oxygenation: A Systematic Review and Meta-Analysis.

OBJECTIVES: Despite the common occurrence of brain injury in patients undergoing extracorporeal membrane oxygenation, it is unclear which cannulation method carries a higher risk of brain injury. We compared the prevalence of brain injury between patients undergoing venoarterial and venovenous extracorporeal membrane oxygenation. DATA SOURCES: PubMed and six other databases from inception to April 2020. STUDY SELECTION: Observational studies and randomized clinical trials in adult patients undergoing venoarterial extracorporeal membrane oxygenation or venovenous extracorporeal membrane oxygenation reporting brain injury. DATA EXTRACTION: Two independent reviewers extracted the data from the studies. Random-effects meta-analyses were used to pool data. DATA SYNTHESIS: Seventy-three studies (n = 16,063) met inclusion criteria encompassing 8,211 patients (51.2%) undergoing venoarterial extracorporeal membrane oxygenation and 7,842 (48.8%) undergoing venovenous extracorporeal membrane oxygenation. Venoarterial extracorporeal membrane oxygenation patients had more overall brain injury compared with venovenous extracorporeal membrane oxygenation (19% vs 10%; p = 0.002). Venoarterial extracorporeal membrane oxygenation patients had more ischemic stroke (10% vs 1%; p < 0.001), hypoxic-ischemic brain injury (13% vs 1%; p < 0.001), and brain death (11% vs 1%; p = 0.001). In contrast, rates of intracerebral hemorrhage (6% vs 8%; p = 0.35) did not differ. Survival was lower in venoarterial extracorporeal membrane oxygenation (48%) than venovenous extracorporeal membrane oxygenation (64%) (p < 0.001). After excluding studies that included extracorporeal cardiopulmonary resuscitation, no significant difference was seen in the rate of overall acute brain injury between venoarterial extracorporeal membrane oxygenation and venovenous extracorporeal membrane oxygenation (13% vs 10%; p = 0.4). However, ischemic stroke (10% vs 1%; p < 0.001), hypoxic-ischemic brain injury (7% vs 1%; p = 0.02), and brain death (9% vs 1%; p = 0.005) remained more frequent in nonextracorporeal cardiopulmonary resuscitation venoarterial extracorporeal membrane oxygenation compared with venovenous extracorporeal membrane oxygenation. CONCLUSIONS: Brain injury was more common in venoarterial extracorporeal membrane oxygenation compared with venovenous extracorporeal membrane oxygenation. While ischemic brain injury was more common in venoarterial extracorporeal membrane oxygenation patients, the rates of intracranial hemorrhage were similar between venoarterial extracorporeal membrane oxygenation and venovenous extracorporeal membrane oxygenation. Further research on mechanism, timing, and effective monitoring of acute brain injury and its management is necessary.

Ischemic Stroke and Intracranial Hemorrhages During Impella Cardiac Support.

Impella is a percutaneously placed, ventricular assist device for short-term cardiac support. We aimed to study acute neurologic complications during short-term cardiac support with Impella. We reviewed prospectively collected data of 79 consecutive persons implanted with Impella at a single tertiary center. Acute neurologic events (ANE) were defined as ischemic strokes or intracranial hemorrhages. Among those with ANE, specific causes of ischemic and hemorrhagic events were collected and discussed. Of 79 persons with Impella with median 8 days of support (range 1-33 days), six (7.5%) developed ANE at a median of 5 days from implant (range 1-8 days). There were three ischemic strokes, two intracerebral hemorrhages, and one subdural hematoma. Hemorrhagic events were attributed to anticoagulant use and thrombocytopenia at the time of the events. Two ischemic strokes were attributed to inadequate anticoagulation with one case of pump thrombosis diagnosed at the time of ischemic stroke. Only two of the six patients survived the acute cardiogenic shock period to achieve heart transplantation. In-hospital ischemic strokes and intracranial hemorrhages are not uncommon during short-term cardiac support period with Impella. Antithrombotic intensity, duration of device support time, and thrombocytopenia might contribute to the incidence of these events.

Brain Injury and Neurologic Outcome in Patients Undergoing Extracorporeal Cardiopulmonary Resuscitation: A Systematic Review and Meta-Analysis.

OBJECTIVES: Extracorporeal cardiopulmonary resuscitation has shown survival benefit in select patients with refractory cardiac arrest but there is insufficient data on the frequency of different types of brain injury. We aimed to systematically review the prevalence, predictors of and survival from neurologic complications in patients who have undergone extracorporeal cardiopulmonary resuscitation. DATA SOURCES: MEDLINE (PubMed) and six other databases (EMBASE, Cochrane Library, CINAHL Plus, Web of Science, and Scopus) from inception to August 2019. STUDY SELECTION: Randomized controlled trials and observational studies in patients greater than 18 years old. DATA EXTRACTION: Two independent reviewers extracted the data. Study quality was assessed by the Cochrane Risk of Bias tool for randomized controlled trials, the Newcastle-Ottawa Scale for cohort and case-control studies, and the Murad tool for case series. Random-effects meta-analyses were used to pool data. DATA SYNTHESIS: The 78 studies included in our analysis encompassed 50,049 patients, of which 6,261 (12.5%) received extracorporeal cardiopulmonary resuscitation. Among extracorporeal cardiopulmonary resuscitation patients, the median age was 56 years (interquartile range, 52-59 yr), 3,933 were male (63%), 3,019 had out-of-hospital cardiac arrest (48%), and 2,289 had initial shockable heart rhythm (37%). The most common etiology of cardiac arrest was acute coronary syndrome (n = 1,657, 50% of reported). The median extracorporeal cardiopulmonary resuscitation duration was 3.2 days (interquartile range, 2.1-4.9 d). Overall, 27% (95% CI, 0.17-0.39%) had at least one neurologic complication, 23% (95% CI, 0.14-0.32%) hypoxic-ischemic brain injury, 6% (95% CI, 0.02-0.11%) ischemic stroke, 6% (95% CI, 0.01-0.16%) seizures, and 4% (95% CI, 0.01-0.1%) intracerebral hemorrhage. Seventeen percent (95% CI, 0.12-0.23%) developed brain death. The overall survival rate after extracorporeal cardiopulmonary resuscitation was 29% (95% CI, 0.26-0.33%) and good neurologic outcome was achieved in 24% (95% CI, 0.21-0.28%). CONCLUSIONS: One in four patients developed acute brain injury after extracorporeal cardiopulmonary resuscitation and the most common type was hypoxic-ischemic brain injury. One in four extracorporeal cardiopulmonary resuscitation patients achieved good neurologic outcome. Further research on assessing predictors of extracorporeal cardiopulmonary resuscitation-associated brain injury is necessary.

Cerebral Microembolization in Left Ventricular Assist Device Associated Ischemic Events.

INTRODUCTION: The significance of microembolic signals (MES) detected by transcranial Doppler ultrasound emboli monitoring (TCD-e) in patients supported with left ventricular assist devices (LVAD) remains unclear. We aimed to investigate the relationship between cerebral microembolization detected by TCD-e and acute ischemic events in LVAD patients. METHODS: We reviewed consecutive patients with acute ischemic stroke or transient ischemic attack (TIA) in a prospectively collected database of LVAD patients. TCD-e exams consisted of monitoring the middle cerebral arteries for microembolic signals (MES) over 30 minutes. RESULTS: Of 515 persons with LVAD, 41 TCD-e studies were performed in 35 patients with acute ischemic stroke or transient ischemic attack (TIA) in a median of 1 day (Interquartile range [IQR]: 0-2) after the event. MES were present in 15 (44%) TCD-e studies with a median MES count of 4 (IQR: 2-15.5). Bloodstream infections were more common in patients with MES (38% versus 8%, P = .039). There were trends for lower international normalized ratio (1.39 versus 1.69, P = .214), lower activated partial thromboplastin (33.2 versus 36.6, P = .577), higher lactate dehydrogenase (531 versus 409, P = .323) and a higher frequency of pump thrombosis (13% versus 8%, P = .637) in patients with MES compared with those without MES. CONCLUSIONS: LVAD patients with acute ischemic stroke or TIA have a high prevalence of MES on TCD-e, which may serve as a marker for a prothrombotic state. Further study of MES in LVAD patients is warranted.

Ultrasound Criteria for Assessment of Vertebral Artery Origins.

BACKGROUND AND PURPOSE: We sought to validate ultrasound as a reliable means of assessing vessel stenosis of vertebral artery origins. METHODS: We reviewed 1,135 patient charts with ultrasound of the posterior circulation performed in 2008-2015 in a single hospital. Inclusion criteria for native vessels consisted of ultrasound and digital subtraction angiography (DSA) performed within 3 months. Patients with indwelling stents were analyzed separately from native vessels. Using DSA as the gold standard, we determined sensitivity and specificity of ultrasound in detecting occlusion at vertebral artery origin. All patients with nonoccluded native vertebral artery origins were evaluated for degree of stenosis on DSA, and compared to mean flow velocity (MFV), peak systolic velocity (PSV), and end-diastolic velocity (EDV) on ultrasound. RESULTS: Among 218 vertebral artery origins in 139 patients evaluated, ultrasound showed sensitivity of 85.7% (95% confidence interval (CI): 69.7-95.2%) for occlusion and specificity of 99.5% (95%CI: 96.9-99.9%). Among 126 arteries without occlusion, <50% stenosis had average MFV (39 ± 19 cm/s), 50-69% stenosis had average MFV (68 ± 35 cm/s), and severe (70-99%) stenosis had average MFV (120 ± 93 cm/s) (P < .001). MFV cutoff value of 44 cm/s corresponded to 77% sensitivity and 70% specificity to detect vertebral artery origin stenosis >50% (C-statistic: .81). PSV value of 97 cm/s corresponded with 72% sensitivity and 70% specificity to detect >50% stenosis (C-statistic: .77). MFV cutoff value of 60 cm/s corresponded with 70% sensitivity and 82% specificity to predict 70-99% stenosis (C-statistic: .83). PSV cutoff value of 110 cm/s corresponded with 80% sensitivity and 72% specificity to predict 70-99% stenosis (C-statistic: .84). CONCLUSION: Ultrasound has good sensitivity and excellent specificity for detecting vertebral origin occlusion. Flow velocity can be used to screen for severe stenosis of vertebral artery at origin.

Clinical course of infectious intracranial aneurysm undergoing antibiotic treatment.

INTRODUCTION: Infectious intracranial aneurysm (IIA, or mycotic aneurysm) is a cerebrovascular complication of infective endocarditis. We aimed to describe the clinical course of IIAs during antibiotic treatment. METHODS: We reviewed medical records of persons with infective endocarditis who underwent cerebral angiography at a single tertiary referral center from 2011 to 2016. Aneurysms were followed with subsequent angiography for unfavorable outcome (growth, rupture, no change, or new IIA formation) or favorable outcome (regression or resolution) until endovascular therapy, aneurysm resolution, or end of observation. RESULTS: Of 618 patients included, 40 (6.5%) had 43 IIAs. Eighteen (42%) aneurysms underwent initial endovascular treatment. Twenty-five unruptured aneurysms were followed for a median 18 antibiotic days after IIA discovery (interquartile range [IQR] 4-32). Eleven (44%) aneurysms had unfavorable outcome (1 rupture, 2 new IIA formation, 6 enlargement, and 2 no change) at median 21 days (IQR 5-32). Favorable angiographic outcome was seen in 7 (28%) patients (6 resolution, 1 regression) at median 36 days (IQR 24-41). Seven aneurysms had no angiographic reevaluations but showed no evidence of rupture during clinical follow-up for median 4 days (IQR 3-12) until hospital discharge. Saccular morphology was associated with unfavorable aneurysmal outcome (p = 0.013). Longer duration of antibiotic exposure prior to IIA discovery was associated with favorable aneurysmal outcome (p = 0.046). CONCLUSION: IIAs represent a dynamic disease. Only a quarter of IIAs resolve with antibiotics alone. Saccular aneurysmal morphology might predict unfavorable aneurysmal outcome. IIA found after longer antibiotic therapy has higher likelihood of resolution or regression on antibiotic treatment.

Early versus Delayed Extracranial-Intracranial Bypass Surgery in Symptomatic Atherosclerotic Occlusion.

BACKGROUND: Clinical trials of extracranial-intracranial (EC-IC) bypass surgery studied patients in subacute and chronic stage after ischemic event. OBJECTIVE: To investigate the short-term outcomes of EC-IC bypass in progressive acute ischemic stroke or recent transient ischemic attacks. METHODS: The study was a retrospective review at a single tertiary referral center from 2008 to 2015. Inclusion criteria consisted of EC-IC bypass within 1 yr of last ischemic symptoms ipsilateral to atherosclerotic occlusion of internal carotid or middle cerebral artery. Early bypass group who underwent surgery within 7 d of last ischemic symptoms were compared to late bypass group who underwent surgery >7 d from last ischemic symptom. The primary endpoint was perioperative ischemic or hemorrhagic stroke or intracranial hemorrhage within 7 d of surgery. RESULTS: Of 126 patients who underwent EC-IC bypass during the period, 81 patients met inclusion criteria, 69 (85%) persons had carotid artery occlusion, 7 (9%) had proximal MCA occlusion, and 5 (6%) had both. Early surgery had a 31% (9/29) perioperative stroke rate compared to 11.5% (6/52) of patients undergoing late bypass (P = .04). Of patients with acute stroke within 7 d of surgery, 41% (7/17) had perioperative stroke within 7 d (P = .07). Six of nine patients (67%) with blood pressure dependent fluctuation of neurologic symptoms had perioperative stroke (P = .049). CONCLUSION: EC-IC bypass in setting of acute symptomatic stroke within 1 wk may confer higher risk of perioperative stroke. Patients undergoing expedited or urgent bypass for unstable or fluctuating stroke symptoms might be at highest risk for perioperative stroke.

MRI Presentation of Infectious Intracranial Aneurysms in Infective Endocarditis.

BACKGROUND: The radiographic appearance of infectious intracranial aneurysms (IIAs) of infective endocarditis (IE) on magnetic resonance imaging (MRI) of brain is varied. We aimed to describe the IIA-specific MRI features in a series of patients with IIAs. METHODS: Records of patients with active IE who had digital subtraction angiography (DSA) at a tertiary medical center from January 2011 to December 2016 were reviewed. MRIs performed prior to IIA treatment were reviewed for findings on susceptibility-weighted imaging (SWI), diffusion-weighted imaging, and T1 with and without contrast. RESULTS: Of the 732 patients with IE, 53 (7%) had IIAs. Of these, 28 patients had an evaluable pre-treatment MRI, in whom 33 IIAs were imaged. MRI to DSA median time was 1 day (interquartile range = 1-5). On MRI, 12 (36%) IIAs had SWI lesion with contrast enhancement, 7 (21%) had cerebral microbleeds, 3 (11%) had sulcal SWI lesion, 2 (6%) IIAs had abscesses, 3 (9%) had intraparenchymal hemorrhage, 3 (9%) had subarachnoid hemorrhage, and 6 (18%) had ischemic stroke at the anatomical locations of IIAs. Four IIAs (12%) had no correlating MRI findings, though those patients had MRI without contrast. CONCLUSION: The MRI features such as SWI lesion and contrast enhancement were the commonest MRI presentations associated with the presence of IIA.

What Causes LVAD-Associated Ischemic Stroke? Surgery, Pump Thrombosis, Antithrombotics, and Infection.

Acute ischemic stroke (AIS) is a major complication in left ventricular assist device (LVAD) population. A better understanding of clinical risk factors associated with AIS may help mitigate risk of stroke. We reviewed prospectively collected data of 477 LVAD patients from a tertiary center from October 1, 2004 to December 31, 2016. Supplemental data abstraction was performed on patients with AIS. Fifty-seven (12%) developed 61 AIS. Of 61, 17 (28%) AIS occurred perioperatively. The median time from implant to perioperative AIS was 5 days (interquartile range: 3-9). Pump thrombosis accounted for 19 (31%) of 61 AIS, and 7 (37%) presented initially with AIS before the pump thrombosis. The median lactate dehydrogenase (LDH) at the time of AIS in the pump thrombosis group (806) was higher than LDH at 1 month (437, P = 0.27) at 3 months (334, P = 0.01), and 6 months (286, P = 0.001) before AIS. Thirty-three (54%) AIS occurred while receiving inadequate antithrombotic therapy. Acute infections were common (31, 51%) in AIS and 12 (20%) were associated with acute bloodstream infection. All AIS were explained by a combination of four clinical risk factors. All LVAD-associated AIS occurred perioperatively or in conjunction with pump thrombosis, subtherapeutic anticoagulation, and bloodstream infection. The common underlying thread is occurrence of a prothrombotic state. The results of this study underscore the potential consequences of disruption of delicate hemostatic balance in patients with LVAD.

The Management of Acute Ischemic Strokes and the Prevalence of Large Vessel Occlusion in Left Ventricular Assist Device.

BACKGROUND: Ischemic and hemorrhagic strokes are frequent complications among those with left ventricular assist device (LVAD). Scarce data exist regarding the prevalence of acute large vessel occlusion (LVO) and treatment of acute ischemic stroke (AIS) in this setting. METHODS: We reviewed prospectively collected data of LVAD patient registry from a single, tertiary center from October 2004 to November 2016. Among those with AIS complications, patients were divided into early stroke (during implantation hospitalization) and late stroke (post-discharge) groups, and neuroimaging was reviewed and data on acute stroke therapy were collected. RESULTS: Of 477 persons with LVAD, 49 (10.3%) AIS occurred. The majority (29/49, 59%) of AIS occurred in-hospital. Thirty-two (65%) persons had international normalized ratios less than 1.7 at the time of AIS, but none qualified to receive acute intravenous thrombolysis. Of 25 (51%) persons who underwent CT angiography (CTA), 33% (16/49) had acute LVOs. Thirty-one percent (5/16) of persons with acute LVOs underwent intra-arterial endovascular therapy. All of 5 cases presented with middle cerebral artery syndrome with a median pre-procedural National Institutes of Health Stroke Scale of 13 (interquartile range 10-18). Successful recanalization was achieved in all 5 cases. CONCLUSIONS: In-hospital strokes and acute LVOs are common in LVAD-associated AIS. Prompt evaluation with CTA and endovascular therapy should be pursued for these critically ill patients.

Structural and functional analysis of an OB-fold in human Ctc1 implicated in telomere maintenance and bone marrow syndromes.

The human CST (Ctc1, Stn1 and Ten1) complex binds the telomeric overhang and regulates telomere length by promoting C-strand replication and inhibiting telomerase-dependent G-strand synthesis. Structural and biochemical studies on the human Stn1 and Ten1 complex revealed its mechanism of assembly and nucleic acid binding. However, little is known about the structural organization of the multi-domain Ctc1 protein and how each of these domains contribute to telomere length regulation. Here, we report the structure of a central domain of human Ctc1. The structure reveals a canonical OB-fold with the two identified disease mutations (R840W and V871M) contributing to the fold of the protein. In vitro assays suggest that although this domain is not contributing directly to Ctc1's substrate binding properties, it affects full-length Ctc1 localization to telomeres and Stn1-Ten1 binding. Moreover, functional assays show that deletion of the entire OB-fold domain leads to significant increase in telomere length, frequency of internal single G-strands and fragile telomeres. Our findings demonstrate that a previously unknown OB-fold domain contributes to efficient Ctc1 telomere localization and chromosome end maintenance.

Magnetic Resonance Imaging Susceptibility-Weighted Imaging Lesion and Contrast Enhancement May Represent Infectious Intracranial Aneurysm in Infective Endocarditis.

BACKGROUND: Infectious intracranial aneurysm (IIA) can complicate infective endocarditis (IE). We aimed to describe the magnetic resonance imaging (MRI) characteristics of IIA. METHODS: We reviewed IIAs among 116 consecutive patients with active IE by conducting a neurological evaluation at a single tertiary referral center from January 2015 to July 2016. MRIs and digital cerebral angiograms (DSA) were reviewed to identify MRI characteristics of IIAs. MRI susceptibility weighted imaging (SWI) was performed to collect data on cerebral microbleeds (CMBs) and sulcal SWI lesions. RESULTS: Out of 116 persons, 74 (63.8%) underwent DSA. IIAs were identified in 13 (17.6% of DSA, 11.2% of entire cohort) and 10 patients with aneurysms underwent MRI with SWI sequence. Nine (90%) out of 10 persons with IIAs had CMB >5 mm or sulcal lesions in SWI (9 in sulci, 6 in parenchyma, and 5 in both). Five out of 8 persons who underwent MRI brain with contrast had enhancement within the SWI lesions. In a multivariate logistic regression analysis, both sulcal SWI lesions (p < 0.001, OR 69, 95% CI 7.8-610) and contrast enhancement (p = 0.007, OR 16.5, 95% CI 2.3-121) were found to be significant predictors of the presence of IIAs. CONCLUSIONS: In the individuals with IE who underwent DSA and MRI, we found that neuroimaging characteristics, such as sulcal SWI lesion with or without contrast enhancement, are associated with the presence of IIA.

Structural and functional analysis of the human POT1-TPP1 telomeric complex.

POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1-TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1-TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1-TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1-TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.

Structural Analysis Reveals the Deleterious Effects of Telomerase Mutations in Bone Marrow Failure Syndromes.

Naturally occurring mutations in the ribonucleoprotein reverse transcriptase, telomerase, are associated with the bone marrow failure syndromes dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis. However, the mechanism by which these mutations impact telomerase function remains unknown. Here we present the structure of the human telomerase C-terminal extension (or thumb domain) determined by the method of single-wavelength anomalous diffraction to 2.31 Å resolution. We also used direct telomerase activity and nucleic acid binding assays to explain how naturally occurring mutations within this portion of telomerase contribute to human disease. The single mutations localize within three highly conserved regions of the telomerase thumb domain referred to as motifs E-I (thumb loop and helix), E-II, and E-III (the FVYL pocket, comprising the hydrophobic residues Phe-1012, Val-1025, Tyr-1089, and Leu-1092). Biochemical data show that the mutations associated with dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis disrupt the binding between the protein subunit reverse transcriptase of the telomerase and its nucleic acid substrates leading to loss of telomerase activity and processivity. Collectively our data show that although these mutations do not alter the overall stability or expression of telomerase reverse transcriptase, these rare genetic disorders are associated with an impaired telomerase holoenzyme that is unable to correctly assemble with its nucleic acid substrates, leading to incomplete telomere extension and telomere attrition, which are hallmarks of these diseases.

Structure and function of the telomeric CST complex.

Telomeres comprise the ends of eukaryotic chromosomes and are essential for cell proliferation and genome maintenance. Telomeres are replicated by telomerase, a ribonucleoprotein (RNP) reverse transcriptase, and are maintained primarily by nucleoprotein complexes such as shelterin (TRF1, TRF2, TIN2, RAP1, POT1, TPP1) and CST (Cdc13/Ctc1, Stn1, Ten1). The focus of this review is on the CST complex and its role in telomere maintenance. Although initially thought to be unique to yeast, it is now evident that the CST complex is present in a diverse range of organisms where it contributes to genome maintenance. The CST accomplishes these tasks via telomere capping and by regulating telomerase and DNA polymerase alpha-primase (polα-primase) access to telomeres, a process closely coordinated with the shelterin complex in most organisms. The goal of this review is to provide a brief but comprehensive account of the diverse, and in some cases organism-dependent, functions of the CST complex and how it contributes to telomere maintenance and cell proliferation.