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.

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.

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.

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.

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.

Structural Basis of Telomerase Inhibition by the Highly Specific BIBR1532.

BIBR1532 is a highly specific telomerase inhibitor, although the molecular basis for inhibition is unknown. Here we present the crystal structure of BIBR1532 bound to Tribolium castaneum catalytic subunit of telomerase (tcTERT). BIBR1532 binds to a conserved hydrophobic pocket (FVYL motif) on the outer surface of the thumb domain. The FVYL motif is near TRBD residues that bind the activation domain (CR4/5) of hTER. RNA binding assays show that the human TERT (hTERT) thumb domain binds the P6.1 stem loop of CR4/5 in vitro. hTERT mutations of the FVYL pocket alter wild-type CR4/5 binding and cause telomere attrition in cells. Furthermore, the hTERT FVYL mutations V1025F, N1028H, and V1090M are implicated in dyskeratosis congenita and aplastic anemia, further supporting the biological and clinical relevance of this novel motif. We propose that CR4/5 contacts with the telomerase thumb domain contribute to telomerase ribonucleoprotein assembly and promote enzymatic activity.