Structural Relationships to Efficacy for Prazole-Derived Antivirals.

Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the ß-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.

Observation of pH-Dependent Residual Structure in the Pmel17 Repeat Domain and the Implication for Its Amyloid Formation.

The varying conformational states of amyloid-forming protein monomers can determine their fibrillation outcome. In this study, we utilize solution NMR and the paramagnetic relaxation enhancement (PRE) effect to observe monomer properties of the repeat domain (RPT) from a human functional amyloid, premelanosomal protein, Pmel17. After excision from the full-length protein, RPT can self-assemble into amyloid fibrils, functioning as a scaffold for melanin deposition. Here, we report possible conformational states of the short RPT (sRPT) isoform, which has been demonstrated to be a fibrillation nucleator. NMR experiments were performed to determine conformational differences in sRPT by comparing aggregation-prone vs nonaggregating solution conditions. We observed significant chemical shift perturbations localized to residues near the C-terminus, demonstrating that the local chemical environment of the amyloid core region is highly sensitive to changes in pH. Next, we introduced cysteine point mutations for the covalent attachment of PRE ligands to sRPT to facilitate the observation of intramolecular interactions. We also utilized solvent PRE molecules with opposing charges to measure changes in the electrostatic potential of sRPT in different pH environments. These observed PRE effects offer insight into initial molecular events that might promote intermolecular interactions, which can trigger fibrillation. Taken together, our results show that sRPT monomers adopt a conformation inconsistent with a fully random coil at neutral pH and undergo conformational changes at lower pH values. These observations highlight regulatory mechanisms via organelle-associated pH conditions that can affect the fibrillation activity of proteins like RPT.

HECT domain interaction with ubiquitin binding sites on Tsg101-UEV controls HIV-1 egress, maturation, and infectivity.

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.

Office- and Bedside-based Screening for Cognitive Impairment and the Dementias: Which Tools to Use, Interpreting the Results, and What Are the Next Steps?

Elderly patients and their families are concerned about the patients' cognitive abilities, and cognitive screening is an efficient diagnostic tool, as long as clinicians administer the screens in a standardized manner and interpret the screen results accurately. The following brief summary reviews commonly used screening instruments and provides information about how to interpret screening test results. It concludes by showing how cognitive screening fits into a four-step process (Education, Screening, Follow-up, and Referral) of how to respond to patients with cognitive concerns.

Secondary thalamic atrophy related to brain infarction may contribute to post-stroke cognitive impairment.

BACKGROUND AND PURPOSE: The thalamus is a key brain hub that is globally connected to many cortical regions. Previous work highlights thalamic contributions to multiple cognitive functions, but few studies have measured thalamic volume changes or cognitive correlates. This study investigates associations between thalamic volumes and post-stroke cognitive function. METHODS: Participants with non-thalamic brain infarcts (3-42 months) underwent MRI and cognitive testing. Focal infarcts and thalami were traced manually. In cases with bilateral infarcts, the side of the primary infarct volume defined the hemisphere involved. Brain parcellation and volumetrics were extracted using a standardized and previously validated neuroimaging pipeline. Age and gender-matched healthy controls provided normal comparative thalamic volumes. Thalamic atrophy was considered when the volume exceeded 2 standard deviations greater than the controls. RESULTS: Thalamic volumes ipsilateral to the infarct in stroke patients (n=55) were smaller than left (4.4 ± 1.4 vs. 5.4 ± 0.5 cc, p < 0.001) and right (4.4 ± 1.4 vs. 5.5 ± 0.6 cc, p < 0.001) thalamic volumes in the controls. After controlling for head-size and global brain atrophy, infarct volume independently correlated with ipsilateral thalamic volume (ß= -0.069, p=0.024). Left thalamic atrophy correlated significantly with poorer cognitive performance (ß = 4.177, p = 0.008), after controlling for demographics and infarct volumes. CONCLUSIONS: Our results suggest that the remote effect of infarction on ipsilateral thalamic volume is associated with global post-stroke cognitive impairment.

Incorporation of residual chemical shift anisotropy into the treatment of 15N pseudocontact shifts for structural refinement.

Paramagnetic NMR experiments, including the pseudocontact shift experiment, have seen increasing use due to recently developed probes and labeling strategies. The pseudocontact shift experiment can provide valuable intra- or inter-molecular distance and orientation information. However, the use of 1H/13C or 1H/15N PCS data in structure calculations is currently complicated by the contribution of residual chemical shift anisotropy to the 13C or 15N datasets. Here, we present a corrected PCS energy term for the software package Xplor-NIH with the appropriate residual chemical shift anisotropy correction and show its suitability for model refinements of ubiquitin labeled at residue 57 with a Tm-M8-SPy tag. For data taken at 800 MHz, the improvement with the corrected energy term is sufficient to make the quality of the fit for the 15N dataset comparable to that of the 1H dataset, for which no correction is needed. The corrected energy term is expected to become more relevant with increased use of higher field instruments and as new paramagnetic probes with larger magnetic susceptibility tensors continue to be developed.

Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport.

Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram-negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse electron paramagnetic resonance (EPR) measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm toward the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, and they demonstrate the importance of studying outer membrane proteins in their native environment.

Bacteria must obtain nutrients from their surrounding environment in order to survive. In Gram-negative bacteria, proteins in the outer membrane surrounding the cell actively transport carbohydrates and trace nutrients like iron into the cell's interior. Although the structures of many of these transport proteins have been determined, the mechanism they use to move molecules across the membrane is poorly understood. To better understand this process, Nilaweera, Nyenhuis and Cafiso examined the structure of BtuB, a transport protein found in the outer membrane of Escherichia coli that is responsible for absorbing vitamin B₁₂. Previous experiments analyzing the structure of BtuB, and other similar transporters, have been carried out on purified proteins that were extracted from the outer membrane. However, these isolated proteins fail to replicate the transport activity observed in bacterial cells. Nilaweera, Nyenhuis and Cafiso therefore wanted to see how the structure of BtuB changes when it is still enclosed in the membrane of E. coli. This revealed that BtuB undergoes large structural changes when it binds to vitamin B₁₂, suggesting that this is an important part of the transport process. However, when purified BtuB was placed into an artificial membrane, these structural changes did not occur. This indicates that the cellular environment in the bacteria is needed for BtuB to carry out its transport role, and explains why previous experiments using purified proteins struggled to see this structural shift. This work highlights the importance of studying bacterial membrane proteins in their native cell environment. BtuB and similar transporters represent a large family of proteins unique to Gram-negative bacteria that have an impact on human health. Since these proteins are structurally alike, the results of this study may help resolve the transport mechanisms of other proteins, ultimately leading to new ways to control bacterial growth.

Novel Tsg101 Binding Partners Regulate Viral L Domain Trafficking.

Two decades ago, Tsg101, a component of the Endosomal Sorting Complexes Required for Transport (ESCRT) complex 1, was identified as a cellular factor recruited by the human immunodeficiency virus type 1 (HIV-1) to facilitate budding of viral particles assembled at the cell periphery. A highly conserved Pro-(Thr/Ser)-Ala-Pro [P(T/S)AP] motif in the HIV-1 structural polyprotein, Gag, engages a P(T/S)AP-binding pocket in the Tsg101 N-terminal domain. Since the same domain in Tsg101 that houses the pocket was found to bind mono-ubiquitin (Ub) non-covalently, Ub binding was speculated to enhance P(T/S)AP interaction. Within the past five years, we found that the Ub-binding site also accommodates di-Ub, with Lys63-linked di-Ub exhibiting the highest affinity. We also identified small molecules capable of disrupting Ub binding and inhibiting budding. The structural similarity of these molecules, prazoles, to nucleosides prompted testing for nucleic acid binding and led to identification of tRNA as a Tsg101 binding partner. Here, we discuss these recently identified interactions and their contribution to the viral assembly process. These new partners may provide additional insight into the control and function of Tsg101 as well as identify opportunities for anti-viral drug design.

A Primary Care Agenda for Brain Health: A Scientific Statement From the American Heart Association.

A healthy brain is critical for living a longer and fuller life. The projected aging of the population, however, raises new challenges in maintaining quality of life. As we age, there is increasing compromise of neuronal activity that affects functions such as cognition, also making the brain vulnerable to disease. Once pathology-induced decline begins, few therapeutic options are available. Prevention is therefore paramount, and primary care can play a critical role. The purpose of this American Heart Association scientific statement is to provide an up-to-date summary for primary care providers in the assessment and modification of risk factors at the individual level that maintain brain health and prevent cognitive impairment. Building on the 2017 American Heart Association/American Stroke Association presidential advisory on defining brain health that included "Life's Simple 7," we describe here modifiable risk factors for cognitive decline, including depression, hypertension, physical inactivity, diabetes, obesity, hyperlipidemia, poor diet, smoking, social isolation, excessive alcohol use, sleep disorders, and hearing loss. These risk factors include behaviors, conditions, and lifestyles that can emerge before adulthood and can be routinely identified and managed by primary care clinicians.

Ebola virus glycoprotein interacts with cholesterol to enhance membrane fusion and cell entry.

Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.

Native Cell Environment Constrains Loop Structure in the Escherichia coli Cobalamin Transporter BtuB.

The extracellular loops of bacterial outer membrane (OM) transporters are thought to sample a range of conformations in the apo state but to undergo a gating motion and assume a more defined conformation upon the binding of substrate. Here, we use pulse electron paramagnetic resonance to examine the conformations of the extracellular loops of BtuB, the Escherichia coli TonB-dependent vitamin B12 transporter, in whole cells. Unlike previous measurements carried out in vitro, the loops assume well-defined configurations in situ that closely match the in surfo crystal structures. Moreover, there is no evidence that the loops undergo significant gating motions upon the binding of substrate. The results demonstrate that the structure of BtuB is dependent upon an intact native OM environment, in which a critical component is likely to be the extracellular lipopolysaccharide. In general, this work indicates that measurements on OM proteins in reconstituted membrane systems may not reflect the native state of the protein in vivo.

Evidence for the Supramolecular Organization of a Bacterial Outer-Membrane Protein from In Vivo Pulse Electron Paramagnetic Resonance Spectroscopy.

In the outer membrane of Gram-negative bacteria, membrane proteins are thought to be organized into domains or islands that play a role in the segregation, movement, and turnover of membrane components. However, there is presently limited information on the structure of these domains or the molecular interactions that mediate domain formation. In the present work, the Escherichia coli outer membrane vitamin B12 transporter, BtuB, was spin-labeled, and double electron-electron resonance was used to measure the distances between proteins in intact cells. These data together with Monte Carlo simulations provide evidence for the presence of specific intermolecular contacts between BtuB monomers that could drive the formation of string-like oligomers. Moreover, the EPR data provide evidence for the location of the interacting interfaces and indicate that lipopolysaccharide mediates the contacts between BtuB monomers.

Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter.

Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here, we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic-facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

PtdInsP2 and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium.

The Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to PtdIns(4,5)P2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.

Vascular cognitive impairment and dementia.

Vascular contributions to cognitive impairment are receiving heightened attention as potentially modifiable factors for dementias of later life. These factors have now been linked not only to vascular cognitive disorders but also Alzheimer's disease. In this chapter we review 3 related topics that address vascular contributions to cognitive impairment: 1. vascular pathogenesis and mechanisms; 2. neuropsychological and neuroimaging phenotypic manifestations of cerebrovascular disease; and 3. prospects for prevention of cognitive impairment of later life based on cardiovascular and stroke risk modification. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Volumetric magnetic resonance imaging correlates of the National Institute of Neurological Disorders and Stroke-Canadian Stroke Network vascular cognitive impairment neuropsychology protocols.

BACKGROUND: Vascular cognitive impairment (VCI) refers to the entire spectrum of cognitive dysfunction attributable to vascular changes in the brain. The objective of this study is to evaluate magnetic resonance imaging (MRI) correlates of performance on the National Institute of Neurological Disorders and Stroke-Canadian Stroke Network (NINDS-CSN) VCI neuropsychology protocols. METHOD: Fifty ischemic stroke patients and 50 normal elderly persons completed the VCI protocols and MRI. Relationships between the four cognitive domains (executive/activation, language, visuospatial, and memory) and three protocol (60-, 30-, and 5-min) summary scores with MRI measures of volumes of white matter hyperintensities (WMH) and global brain and hippocampal atrophy were assessed using linear regression. RESULTS: All cognitive domain scores were associated with WMH volume and, with the exception of language domain, with global atrophy. Additional relationships were found between executive/activation and language domains with left hippocampal volume, visuospatial domain with right hippocampal volume, and memory domain with bilateral hippocampal volumes. All protocol summary scores showed comparable relationships with WMH and hippocampal volumes, with additional relationships found between the 60- and 30-min protocols with global brain volume. CONCLUSIONS: Performance on the NINDS-CSN VCI protocols reflects underlying volumetric brain changes implicated in cognitive dysfunctions in VCI.

Trail Making Test Elucidates Neural Substrates of Specific Poststroke Executive Dysfunctions.

BACKGROUND AND PURPOSE: Poststroke cognitive impairment is typified by prominent deficits in processing speed and executive function. However, the underlying neuroanatomical substrates of executive deficits are not well understood, and further elucidation is needed. There may be utility in fractionating executive functions to delineate neural substrates. METHODS: One test amenable to fine delineation is the Trail Making Test (TMT), which emphasizes processing speed (TMT-A) and set shifting (TMT-B-A difference, proportion, quotient scores, and TMT-B set-shifting errors). The TMT was administered to 2 overt ischemic stroke cohorts from a multinational study: (1) a chronic stroke cohort (N=61) and (2) an acute-subacute stroke cohort (N=45). Volumetric quantification of ischemic stroke and white matter hyperintensities was done on magnetic resonance imaging, along with ratings of involvement of cholinergic projections, using the previously published cholinergic hyperintensities projections scale. Damage to the superior longitudinal fasciculus, which colocalizes with some cholinergic projections, was also documented. RESULTS: Multiple linear regression analyses were completed. Although larger infarcts (ß=0.37, P<0.0001) were associated with slower processing speed, cholinergic hyperintensities projections scale severity (ß=0.39, P<0.0001) was associated with all metrics of set shifting. Left superior longitudinal fasciculus damage, however, was only associated with the difference score (ß=0.17, P=0.03). These findings were replicated in both cohorts. Patients with ≥2 TMT-B set-shifting errors also had greater cholinergic hyperintensities projections scale severity. CONCLUSIONS: In this multinational stroke cohort study, damage to lateral cholinergic pathways and the superior longitudinal fasciculus emerged as significant neuroanatomical correlates for executive deficits in set shifting.