SKIP controls lysosome positioning using a composite kinesin-1 heavy and light chain-binding domain

The 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 tryptophanacidic- 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

The light chains of kinesin-1 are autoinhibited

The 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

Binding of myomesin to obscurin-like-1 to the muscle M-band provides a strategy for isoform-specific mechanical protection

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signalling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1): myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of 135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.We thank the Diamond Light Source and the European Synchrotron Radiation Laboratory for access to MX and SAXS beamlines, respectively. This work was supported by a British Heart Foundation grant (PG/10/67/28527) awarded to R.A.S. and M.G. as well as MRC grant MR/J010456/1 to M.G. and a British Heart Foundation grant (PG/13/50/30426) and EPSRC Fellowship (K00641X/1) to S.G.-M

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

The 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 (KLC TPR ). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call \u2018Y-acidic\u2019 (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1 TPR 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 KLC TPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity

Nonalcoholic Fatty Liver Disease Is Associated With Ventricular Arrhythmias in Patients With Type 2 Diabetes Referred for Clinically Indicated 24-Hour Holter Monitoring

Recent studies have suggested that nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of heart rate-corrected QT interval prolongation and atrial fibrillation in patients with type 2 diabetes. Currently, no data exist regarding the relationship between NAFLD and ventricular arrhythmias in this patient population

Heart valve calcification in patients with type 2 diabetes and nonalcoholic fatty liver disease

PurposeAortic valve sclerosis (AVS) and mitral annulus calcification (MAC) are two powerful predictors of adverse cardiovascular outcomes in patients with type 2 diabetes, but the aetiology of valvular calcification is uncertain. Nonalcoholic fatty liver disease (NAFLD) is an emerging cardiovascular risk factor and is very common in type 2 diabetes, but whether NAFLD is associated with valvular calcification in this group of patients is presently unknown.MethodsWe undertook a cross-sectional study of 247 consecutive type 2 diabetic outpatients with no previous history of heart failure, valvular heart diseases (aortic stenosis, mitral stenosis, moderate or severe aortic and mitral regurgitation) or hepatic diseases. Presence of MAC and AVS was detected by echocardiography. NAFLD was diagnosed by ultrasonography.ResultsOverall, 139 (56.3%) patients had no heart valve calcification (HVC-0), 65 (26.3%) patients had one valve affected (HVC-1) and 43 (17.4%) patients had both valves affected (HVC-2). 175 (70.8%) patients had NAFLD and the prevalence of this disease markedly increased in patients with HVC-2 compared with either HVC-1 or HVC-0 (86.1% vs. 83.1% vs. 60.4%, respectively; p<0.001). NAFLD was significantly associated with AVS and/or MAC (unadjusted-odds ratio 3.51, 95%CI 1.89–6.51, p<0.001). Adjustments for age, sex, waist circumference, smoking, blood pressure, hemoglobin A1c, LDL-cholesterol, kidney function parameters, medication use and echocardiographic variables did not appreciably weaken this association (adjusted-odds ratio 2.70, 95%CI 1.23-7.38, p<0.01).ConclusionsOur results show that NAFLD is an independent predictor of cardiac calcification in both the aortic and mitral valves in patients with type 2 diabetes

Structural basis for kinesin-1:cargo recognition

Kinesin-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

The light chains of kinesin-1 are autoinhibited

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC(TPR)), 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 KLC2(TPR) 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.</p

American Diabetes Association - 75th Scientific Meeting; Section: Epidemiology/Genetics; Poster n. 1581-P: "Nonalcoholic Fatty Liver Disease Is Associated with Heart Valve Calcification in Type 2 Diabetes"

Aortic valve sclerosis (AVS) and mitral annulus calcifi cation (MAC) are powerfulpredictors of adverse cardiovascular outcomes in patients with type 2diabetes (T2D), but the aetiology of valvular calcifi cation is uncertain. Nonalcoholicfatty liver disease (NAFLD) is an emerging cardiovascular risk factorcommonly present in T2D patients, but its association with valvular calcifi -cation is unknown. We sought to investigate whether NAFLD is associatedwith AVS and/or MAC in T2D patients. We conducted a cross-sectional studyby performing a conventional echocardiography and liver ultrasonography ina sample of 247 consecutive outpatients with T2D (179 men; mean age 68years) free of known liver diseases, prior history of chronic heart failure andmoderate-to-severe valvular heart disease. Overall, 139 (56.3%) patients hadno calcifi cation at both aortic and mitral valve (HVC-0), 65 (26.3%) had onevalve affected (HVC-1) and 43 (17.4%) patients had both valves affected (HVC-2). NAFLD was present in 175 (70.8%) patients and its prevalence markedlyincreased in patients with HVC-2 compared with either HVC-1 or HVC-0 (86.1%vs. 83.1% vs. 60.4%, respectively; p&lt;0.001). NAFLD was associated with AVSand/or MAC (unadjusted-odds ratio [OR] 3.51, 95% CI 1.89-6.51, p&lt;0.001). Adjustmentsfor age, sex, smoking history, alcohol consumption, diastolic bloodpressure, hemoglobin A1c, LDL-cholesterol, estimated glomerular fi ltrationrate, use of hypoglycemic, lipid-lowering and anti-hypertensive medicationsand echocardiographic variables did not substantially attenuate the strong associationof NAFLD with AVS and/or MAC (adjusted-OR 2.97, 95% CI 1.31-6.70,p&lt;0.01). In conclusion, these results show for the fi rst time that NAFLD is astrong and independent predictor of cardiac calcifi cation in both aortic andmitral valves in patients affected by T2D. Further research is needed to betterelucidate the mechanisms underlying this association

STUDIES ON THE EXPLOSION (PART 1) : ON THE PRILLED AMMONIUM NITRATE

We observed prilled ammonium nitrate by the electron and optical microscopes. Wealso measured its bulk density, ratio of oil absorption and the detonation velocity of ~mmoniumnitrate fuel oil blasting agents, I. E. the mixture of the prilled AN and FO. We further madethe cap sensitivity tests and the drop hammer tests.Through these measurings and testings, we concluded that the prilled AN which had thefollowing properties was the most suitable for ANFO blasting agents; bulk density: 0.75 .-0.85g/cc, ratio of oil absorption: IO-2Og/ 100 g AN, water content: 0.5% or below, detonationvelocity: 2, 500-3, 300m 'sec, cap sensitivity: unable to be detonated by a No.8 cap.We observed prilled ammonium nitrate by the electron and optical microscopes. Wealso measured its bulk density, ratio of oil absorption and the detonation velocity of ~mmoniumnitrate fuel oil blasting agents, I. E. the mixture of the prilled AN and FO. We further madethe cap sensitivity tests and the drop hammer tests.Through these measurings and testings, we concluded that the prilled AN which had thefollowing properties was the most suitable for ANFO blasting agents; bulk density: 0.75 .-0.85g/cc, ratio of oil absorption: IO-2Og/ 100 g AN, water content: 0.5% or below, detonationvelocity: 2,500-3,300m 'sec, cap sensitivity: unable to be detonated by a No.8 cap