Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure.

We have searched for a minimal interaction motif in tau protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of tau plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length tau.

A new look at the microtubule binding patterns of dimeric kinesins.

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations.

Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent.

Background: Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases.

Results: The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1.

Crystallization of a macromolecular ring assembly of tubulin liganded with the anti-mitotic drug podophyllotoxin.

The interaction of the anti-cancer drug podophyllotoxin with a high-molecular-weight assembly of tubulin has been employed to produce three-dimensional crystals from avian erythrocyte tubulin as well as from pig brain tubulin. Avian erythrocyte tubulin crystals belong to the space group C2 with unit cell dimensions a = 740 A, b = 330 A, c = 460 A, beta = 128 degrees. The basis of these crystals is ring oligomers with a molecular mass of approximately 6 x 10(6) Da.

Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells.

Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro.

The structure of the nucleotide-binding site of kinesin.

Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(-)4, and (3) with ADP hydrolyzed to AMP by apyrase.

Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain.

Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing.

Tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles.

We have performed a real time analysis of fluorescence-tagged vesicle and mitochondria movement in living CHO cells transfected with microtubule-associated protein tau or its microtubule-binding domain. tau does not alter the speed of moving vesicles, but it affects the frequencies of attachment and detachment to the microtubule tracks. Thus, tau decreases the run lengths both for plus-end and minus-end directed motion to an equal extent.

Assembly of paired helical filaments from mouse tau: implications for the neurofibrillary pathology in transgenic mouse models for Alzheimer's disease.

In Alzheimer's disease and related dementias, human tau protein aggregates into paired helical filaments and neurofibrillary tangles. However, such tau aggregates have not yet been demonstrated in transgenic mouse models of the disease. One of the possible explanations would be that mouse tau has different properties which prevents it from aggregating.

Motor proteins of the kinesin family. Structures, variations, and nucleotide binding sites.

Microtubule-dependent motors of the kinesin family convert the energy from ATP hydrolysis into mechanical work in order to transport vesicles and organelles along microtubules. The motor domains of several kinesins have been solved by X-ray diffraction, but the conformational changes associated with force development remain unknown. Here we describe conformational properties of kinesin that might be related to the mechanism of action.

Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments.

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs.

The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains.

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala.

Structures of kinesin and kinesin-microtubule interactions.

Several X-ray crystal structures of kinesin motor domains have recently been solved at high resolution ( approximately 0.2-0.3 nm), in both their monomeric and dimeric states.

A nucleated assembly mechanism of Alzheimer paired helical filaments.

Alzheimer's disease is characterized by two types of fibrous aggregates in the affected brains, the amyloid fibers (consisting of the Abeta-peptide, generating the amyloid plaques), and paired helical filaments (PHFs; made up of tau protein, forming the neurofibrillary tangles). Hence, tau protein, a highly soluble protein that normally stabilizes microtubules, becomes aggregated into insoluble fibers that obstruct the cytoplasm of neurons and cause a loss of microtubule stability. We have developed recently a rapid assay for monitoring PHF assembly and show here that PHFs arise from a nucleated assembly mechanism.

Tau in Alzheimer's disease.

Neurofibrillar protein aggregates containing tau are one of the major hallmarks of Alzheimer's disease (AD). In normal cells, tau stabilizes axonal microtubules, which are the tracks for intracellular traffic. In AD, tau becomes abnormally phosphorylated, aggregates into paired helical filaments and loses its ability to maintain the microtubule tracks.

The structural and mechanochemical cycle of kinesin.

Kinesin is a microtubule-based motor protein that pulls vesicles or organelles towards the plus end of microtubules. Structural changes in the protein that drive motility are coupled to ATP binding and hydrolysis. Here, we attempt to integrate recent structural and kinetic results into a picture of the mechanochemical cycle of kinesin.

Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease.

The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein-driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins.

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules.

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al.

MAPs, MARKs and microtubule dynamics.

Microtubules (MTs) serve as tracks for cellular transport, and regulate cell shape and polarity. Rapid transitions between stable and dynamic forms of MTs are central to these processes. This dynamic instability is regulated by a number of cellular factors, including the structural MT-associated proteins (MAPs), which in turn are regulated by phosphorylation.

The coiled-coil helix in the neck of kinesin.

Kinesin is a microtubule-dependent motor protein. We have recently determined the X-ray structure of monomeric and dimeric kinesin from rat brain. The dimer consists of two motor domains, held together by their alpha-helical neck domains forming a coiled coil.

Mitotic phosphorylation of tau protein in neuronal cell lines resembles phosphorylation in Alzheimer's disease.

Tau protein, a neuronal microtubule-associated protein is phosphorylated on several sites when extracted from brain tissue and is a substrate for many protein kinases in vitro. In Alzheimer's disease it becomes hyperphosphorylated, notably at Ser-Pro or Thr-Pro motifs, and forms the paired helical filaments (PHFs). The increased phosphorylation can be detected by several antibodies raised against Alzheimer tau.

Histological markers in nasal mucosa of patients with Alzheimer's disease.

Neuropathological changes such as dystrophic neurites and the presence of abnormal tau protein in the olfactory system, including primary sensory cells and nerve fibres have previously been demonstrated in nasal mucosa tissue of patients with Alzheimer's disease (AD). These changes were detected in autopsy-derived material from histopathologically confirmed AD cases as well as in biopsy tissue from clinical severely ill AD patients. To investigate the potential usefulness for the early diagnosis of AD, we obtained biopsy tissue from olfactory mucosa from 5 clinically mild to moderate AD patients and stained for the presence of tau or beta-amyloid by immunocytochemistry using a panel of specific antibodies.

Rapid assembly of Alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution.

Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consisting of the Abeta peptide, generating the amyloid plaques) and paired helical filaments (PHFs, made up of tau protein, forming the neurofibrillary tangles). While the principles of amyloid aggregation are known in some detail, the investigation of PHF assembly has been hampered by the low efficiency of tau aggregation, the requirement of high protein concentrations, and the lack of suitable detection methods. Here we report a quantitative assay system that permits monitoring of the assembly of PHFs in real time by the fluorescence of dyes such as thioflavine S or T.