62,852 research outputs found
Structural basis for kinesin-1:cargo recognition
No full textKinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here, we present the crystal structure of the tetratricopeptide repeat domain of kinesin light chain 2 in complex with a cargo peptide harboring a "tryptophan-acidic" motif derived from SKIP (SifA-kinesin interacting protein), a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical, and cellular assays allow us to propose a framework for intracellular transport based on the binding by kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for kinesin-1:cargo recognition
Fragment-linking peptide design yields a high-affinity ligand for microtubule-based transport
No full textSynthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides which make improved interactions with the target. Here, we devise a fragment-linking strategy - ‘mash-up’ design - to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolaraffinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.<br/
The light chains of kinesin-1 are autoinhibited
No full textThe light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLCTPR), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme
SKIP controls lysosome positioning using a composite kinesin-1 heavy and light chain-binding domain
No full textThe molecular interplay between cargo recognition and regulation of the activity of the kinesin-1 microtubule motor is not well understood. Using the lysosome adaptor SKIP (also known as PLEKHM2) as model cargo, we show that the kinesin heavy chains (KHCs), in addition to the kinesin light chains (KLCs), can recognize tryptophan-acidic-binding determinants on the cargo when presented in the context of an extended KHC-interacting domain. Mutational separation of KHC and KLC binding shows that both interactions are important for SKIP-kinesin-1 interaction in vitro and that KHC binding is important for lysosome transport in vivo However, in the absence of KLCs, SKIP can only bind to KHC when autoinhibition is relieved, suggesting that the KLCs gate access to the KHCs. We propose a model whereby tryptophan-acidic cargo is first recognized by KLCs, resulting in destabilization of KHC autoinhibition. This primary event then makes accessible a second SKIP-binding site on the KHC C-terminal tail that is adjacent to the autoinhibitory IAK region. Thus, cargo recognition and concurrent activation of kinesin-1 proceed in hierarchical stepwise fashion driven by a dynamic network of inter- and intra-molecular interactions.</p
Changes in alcohol history taking and management of alcohol dependence by interns at The Royal Adelaide Hospital
No full textObjectiveThe objective of this study is to determine whether the presence of a drug and alcohol unit and comprehensive medical education have made a difference to the detection and management of alcohol-related problems in a general teaching hospital.MethodsData were obtained from hospital case notes before and after the introduction of the drug and alcohol unit and medical education. Samples of general admissions and admissions with an alcohol diagnosis were obtained. The outcome measures included alcohol history taking, quantification of alcohol consumption and management of alcohol dependence.ResultsIt was found that for the general admission sample, interns were more likely to take an alcohol history, both in the accident and emergency (A&E) department and on the ward, in 1994 compared to 1988. The difference in the A&E department was statistically significant (P = 0.009). In the A&E department in 1994 there was a greater prevalence of alcohol history taking for male patients (odds ratio (OR) 10.09, 95% confidence interval (CI) 1.89 to 53.70, P = 0.007) compared with female patients (OR = 1.81, 95% CI 0.38 to 8.57, P = 0.045). There were no differences in alcohol history taking by interns in the samples of alcohol-related admissions. There were no statistically significant differences in the prevalence of documenting alcohol histories quantitatively in either sample. The use of alcohol withdrawal charts, ordering diazepam for alcohol withdrawal and ordering drugs which conformed to the hospital formulary all increased significantly by 1994. The prevalence of ordering thiamine and consulting the drug and alcohol unit both decreased slightly by 1994, but not significantly.ConclusionImprovements have been found in the detection and management of alcohol use since the introduction of the Drug and Alcohol Unit and medical education, but there is still room for further improvement and particular areas where greater attention is needed are identified.Matt Gaughwin, Jane Dodding, Jason M White, Philip Rya
The Dynamic Localization of Cytoplasmic Dynein in Neurons Is Driven by Kinesin-1
No full textCytoplasmic dynein, the major motor driving retrograde axonal transport, must be actively localized to axon terminals. This localization is critical as dynein powers essential retrograde trafficking events required for neuronal survival, such as neurotrophic signaling. Here, we demonstrate that the outward transport of dynein from soma to axon terminal is driven by direct interactions with the anterograde motor kinesin-1. In developing neurons, we find that dynein dynamically cycles between neurites, following kinesin-1 and accumulating in the nascent axon coincident with axon specification. In established axons, dynein is constantly transported down the axon at slow axonal transport speeds; inhibition of the kinesin-1-dynein interaction effectively blocks this process. In vitro and live-imaging assays to investigate the underlying mechanism lead us to propose a new model for the slow axonal transport of cytosolic cargos, based on short-lived direct interactions of cargo with a highly processive anterograde motor
A 2 h periodic variation in the low-mass X-ray binary Ser X-1
No full textSpectroscopy of the low-mass X-ray binary Ser X-1 using the Gran Telescopio Canarias have revealed a ?2 h periodic variability that is present in the three strongest emission lines. We tentatively interpret this variability as due to orbital motion, making it the first indication of the orbital period of Ser X-1. Together with the fact that the emission lines are remarkably narrow, but still resolved, we show that a main-sequence K dwarf together with a canonical 1.4 M? neutron star gives a good description of the system. In this scenario, the most likely place for the emission lines to arise is the accretion disc, instead of a localized region in the binary (such as the irradiated surface or the stream-impact point), and their narrowness is due instead to the low inclination (?10°) of Ser X-1
Stable voters in an unstable party environment : continuity and change in Italian electoral behaviour
No full textM.24981-1999 Paolo Segatti, Paolo Bellucci and Marco Maraffi. 30 cm. A previous version of this paper was presented at a symposium on Political Parties : Changing Roles in Contemporary Democracies, held at the Center for Advanced Study in the Social Sciences of the Juan March Institute, Madrid, December 15-17, 1994. -- P.1. Includes bibliographical references (p. 56-59
Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors
No full textThe light chains (KLCs) of the heterotetrameric microtubule motor kinesin-1, that bind to cargo adaptor proteins and regulate its activity, have a capacity to recognize short peptides via their tetratricopeptide repeat domains (KLCTPR). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call 'Y-acidic' (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1TPR are distinct from those utilized for the recognition of W-acidic motifs found in adaptors that are KLC- isoform non-selective. However, a partial overlap on their receptor binding sites implies that adaptors relying on Y-acidic and W-acidic motifs must act independently. We propose a model to explain why these two classes of motifs that bind to the concave surface of KLCTPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity
De Maiestate / Praeside M. Jacobo Thomasio, Moralis Philosoph. P. P., publice disputabit Johannes Dunte, R. L. Author & Respon: ad diem 9. Septembr. H L. Q. C.
No full textDE MAIESTATE / PRAESIDE M. JACOBO THOMASIO, MORALIS PHILOSOPH. P. P., PUBLICE DISPUTABIT JOHANNES DUNTE, R. L. AUTHOR & RESPON: AD DIEM 9. SEPTEMBR. H L. Q. C.
De Maiestate / Praeside M. Jacobo Thomasio, Moralis Philosoph. P. P., publice disputabit Johannes Dunte, R. L. Author & Respon: ad diem 9. Septembr. H L. Q. C. (1)
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