Reversible lysine fatty acylation of an anchoring protein mediates adipocyte adrenergic signaling.

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase-anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via ß2- and ß3-adrenergic receptors (ß-ARs), which are G protein-coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives ß-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes.

The production effect is consistent over material variations: support for the distinctiveness account.

The production effect is the superior memory for items read aloud as opposed to silently at the time of study. The distinctiveness account holds that produced items benefit from the encoding of additional elements associated with the act of production. If so, then that benefit should be consistent regardless of item type. Three experiments, using three different sets of materials and three different methods, tested this hypothesis. Experiment 1, using recognition testing, showed consistent production benefits for high and low frequency words. Experiment 2, using free recall, showed consistent production increments for pictures and words. Experiment 3, using incidental learning, showed consistent production benefits for recognition of nonwords and words. Taken together, these results fit with the distinctiveness account: Production at encoding dependably adds information to the memory record, regardless of item type or method of testing, producing a consistently reliable memory benefit.

Uptake of high-density lipoprotein by scavenger receptor class B type 1 is associated with prostate cancer proliferation and tumor progression in mice.

High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however, its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts than SR-B1+/+ cells in the same host model. Increased tumor volume was overall associated with reduced survival. We conclude that knocking out SR-B1 in prostate cancer tumors reduces HDL-associated increases in prostate cancer cell proliferation and disease progression.

A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function.

Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these-acetylation of lysine 146 in Eg5-by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site-neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant-features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a "brake" that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.

Beyond the genome: challenges and potential for epigenetics-driven therapeutic approaches in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease of the cardiopulmonary system caused by the narrowing of the pulmonary arteries, leading to increased vascular resistance and pressure. This leads to right ventricle remodeling, dysfunction, and eventually, death. While conventional therapies have largely focused on targeting vasodilation, other pathological features of PAH including aberrant inflammation, mitochondrial dynamics, cell proliferation, and migration have not been well explored. Thus, despite some recent improvements in PAH treatment, the life expectancy and quality of life for patients with PAH remains poor. Showing many similarities to cancers, PAH is characterized by increased pulmonary arterial smooth muscle cell proliferation, decreased apoptotic signaling pathways, and changes in metabolism. The recent successes of therapies targeting epigenetic modifiers for the treatment of cancer has prompted epigenetic research in PAH, revealing many new potential therapeutic targets. In this minireview we discuss the emergence of epigenetic dysregulation in PAH and highlight epigenetic-targeting compounds that may be effective for the treatment of PAH.

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5.

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction--including switch-1 and the neck linker--how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as "structural kinetics," to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.

Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family.

Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.

Cross Section of Legislative Approaches to Reducing Indoor Dampness and Mold.

Exposure to indoor dampness and mold is associated with numerous adverse respiratory conditions, including asthma. While no quantitative health-based threshold currently exists for mold, the conditions that support excessive dampness and mold are known and preventable; experts agree that controlling these conditions could lead to substantial savings in health care costs and improvement in public health. This article reviews a sample of state and local policies to limit potentially harmful exposures. Adoption of laws to strengthen building codes, specify dampness and mold in habitability laws, regulate mold contractors, and other legislative approaches are discussed, as are key factors supporting successful implementation. Communicating these lessons learned could accelerate the process for other jurisdictions considering similar approaches. Information about effectiveness of legislation as prevention is lacking; thus, evaluation could yield important information to inform the development of model state or local laws that significantly address mold as a public health concern.

Interplay between the E2F pathway and ß-adrenergic signaling in the pathological hypertrophic response of myocardium.

The E2F/Pocket protein (Rb) pathway regulates cell growth, differentiation, and death by modulating gene expression. We previously examined this pathway in the myocardium via manipulation of the unique E2F repressor, E2F6, which is believed to repress gene activity independently of Rb. Mice with targeted expression of E2F6 in postnatal myocardium developed dilated cardiomyopathy (DCM) without hypertrophic growth. We assessed the mechanisms of the apparent failure of compensatory hypertrophic growth as well as their response to the ß-adrenergic agonist isoproterenol. As early as 2 weeks, E2F6 transgenic (Tg) mice present with dilated thinner left ventricles and significantly reduced ejection fraction and fractional shortening which persists at 6 weeks of age, but with no apparent increase in left ventricle weight: body weight (LVW:BW). E2F6-Tg mice treated with isoproterenol (6.1 mg/kg/day) show double the increase in LVW:BW than their Wt counterparts (32% vs 16%, p-value: 0.007). Western blot analysis revealed the activation of the adrenergic pathway in Tg heart tissue under basal conditions with ~2-fold increase in the level of ß2-adrenergic receptors (p-value: 8.9E-05), protein kinase A catalytic subunit (PKA-C) (p-value: 0.0176), activated c-Src tyrosine-protein kinase (p-value: 0.0002), extracellular receptor kinase 2 (ERK2) (p-value: 0.0005), and induction of the anti-apoptotic protein Bcl2 (p-value 0. 0.00001). In contrast, a ~60% decrease in the cardiac growth regulator: AKT1 (p-value 0.0001) and a ~four fold increase in cyclic AMP dependent phosphodiesterase 4D (PDE4D), the negative regulator of PKA activity, were evident in the myocardium of E2F6-Tg mice. The expression of E2F3 was down-regulated by E2F6, but was restored by isoproterenol. Further, Rb expression was down-regulated in Tg mice in response to isoproterenol implying a net activation of the E2F pathway. Thus the unique regulation of E2F activity by E2F6 renders the myocardium hypersensitive to adrenergic stimulus resulting in robust hypertrophic growth. These data reveal a novel interplay between the E2F pathway, ß2-adrenergic/PKA/PDE4D, and ERK/c-Src axis in fine tuning the pathological hypertrophic growth response. E2F6 deregulates E2F3 such that pro-hypertrophic growth and survival are enhanced via ß2-adrenergic signaling however this response is outweighed by the induction of anti-hypertrophic signals so that left ventricle dilation proceeds without any increase in muscle mass.

"Snapshots" of ispinesib-induced conformational changes in the mitotic kinesin Eg5.

Kinesins comprise a superfamily of molecular motors that drive a wide variety of cellular physiologies, from cytoplasmic transport to formation of the bipolar spindle in mitosis. These differing roles are reflected in corresponding polymorphisms in key kinesin structural elements. One of these is a unique loop and stem motif found in all kinesins and referred to as loop 5 (L5). This loop is longest in the mitotic kinesin Eg5 and is the target for a number of small molecule inhibitors, including ispinesib, which is being used in clinical trials in patients with cancer. In this study, we have used x-ray crystallography to identify a new structure of an Eg5-ispinesib complex and have combined this with transient state kinetics to identify a plausible sequence of conformational changes that occur in response to ispinesib binding. Our results demonstrate that ispinesib-induced structural changes in L5 from Eg5 lead to subsequent changes in the conformation of the switch II loop and helix and in the neck linker. We conclude that L5 in Eg5 simultaneously regulates the structure of both the ATP binding site and the motor's mechanical elements that generate force.

Loop L5 assumes three distinct orientations during the ATPase cycle of the mitotic kinesin Eg5: a transient and time-resolved fluorescence study.

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.

Remembered study mode: support for the distinctiveness account of the production effect.

The production effect is the finding that words spoken aloud at study are subsequently remembered better than are words read silently at study. According to the distinctiveness account, aloud words are remembered better because the act of speaking those words aloud is encoded and later recovery of this information can be used to infer that those words were studied. An alternative account (the strength-based account) is that memory strength is simply greater for words read aloud. To discriminate these two accounts, we investigated study mode judgements (i.e., "aloud"/"silent"/"new" ratings): The strength-based account predicts that "aloud" responses should positively correlate with memory strength, whereas the distinctiveness account predicts that accuracy of study mode judgements will be independent of memory strength. Across three experiments, where the strength of some silent words was increased by repetition, study mode was discriminable regardless of strength-even when the strength of aloud and repeated silent items was equivalent. Consistent with the distinctiveness account, we conclude that memory for "aloudness" is independent of memory strength and a likely candidate to explain the production effect.

Consolidated Principles for Equitable and Inclusive Digital Health and Virtual Care Co-Design.

Digital health and virtual care (DH/VC) interventions have been rapidly transforming healthcare systems, offering enormous potential to bridge gaps in healthcare access and deliver person-centred interventions to equity-deserving populations. Working in partnership with patients, caregivers and communities to meaningfully integrate lived experience perspectives into DH/VC interventions can help ensure that diverse needs are met. In this commentary, we propose a consolidated set of principles for co-designing equity-informed DH/VC interventions. We also identify how these principles can be leveraged through resources and opportunities offered by Healthcare Excellence Canada and others.

The structural basis of force generation by the mitotic motor kinesin-5.

Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.

Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state.

Platelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbßIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.

The E2F6 repressor activates gene expression in myocardium resulting in dilated cardiomyopathy.

The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P<0.05) in left ventricular end-diastolic diameter and reduced ejection fraction (<40%, P<0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F-responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P<0.01) decrease in gap junction protein connexin-43, resulting in abnormal electrocardiogram including a 24% (P<0.05) increase in PR interval. Further, mir-206, which targets connexin-43, was up-regulated 10-fold (P<0.05) in Tg myocardium. The mitogen-activated protein kinase pathway, which regulates the levels of miR-206 and connexin-43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post-transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.

HDAC11 inhibition triggers bimodal thermogenic pathways to circumvent adipocyte catecholamine resistance.

Stimulation of adipocyte ß-adrenergic receptors (ß-ARs) induces expression of uncoupling protein 1 (UCP1), promoting nonshivering thermogenesis. Association of ß-ARs with a lysine-myristoylated form of A kinase-anchoring protein 12 (AKAP12, also known as gravin-α) is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of ß-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a noncanonical acute response that is posttranscriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where ß-AR signaling is blocked. These findings define cell-autonomous, multimodal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of ß-AR stimulation.

HDAC11 inhibition triggers bimodal thermogenic pathways to circumvent adipocyte catecholamine resistance.

Stimulation of adipocyte ß-adrenergic receptors (ß-ARs) induces expression of uncoupling protein 1 (UCP1), promoting non-shivering thermogenesis. Association of ß-ARs with a lysine myristoylated form of A-kinase anchoring protein 12 (AKAP12)/gravin-α is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of ß-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a non-canonical acute response that is post-transcriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where ß-AR signaling is blocked. These findings define cell autonomous, multi-modal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of ß-AR stimulation.

The kinesin-5 tail and bipolar minifilament domains are the origin of its microtubule crosslinking and sliding activity.

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.