János Kovács

Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies

The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinsons (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Picks disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimers disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation.

Recently, we isolated from bovine brain a protein, TPPP/p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via blast. Here, we characterized the binding of TPPP/p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 × 104 M–1·s–1; koff, 5.4 × 10–3 s–1; and Kd, 2.3 × 10–7 M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP/p25 into cleavage Drosophila embryos expressing tubulin–GFP fusion protein reveals that TPPP/p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP/p25 both in vitro and in vivo.

Interactions of Pathological Hallmark Proteins TUBULIN POLYMERIZATION PROMOTING PROTEIN/p25, β-AMYLOID, AND α-SYNUCLEIN

Background: The disordered TPPP/p25 is a hallmark of synucleinopathies. Results: Tight binding of TPPP/p25 with β-amyloid results in the formation of massive aggregates both in vitro and in vivo. Conclusion: The presence of intracellular pathological-like TPPP/p25-β-amyloid aggregates elucidates the partial co-localization of β-amyloid and TPPP/p25 in Lewy body dementia with Alzheimer disease. Significance: This new type of aggregation may form bridge to conjoin synucleopathies with other neuropathologies. The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ42 tightly bound to TPPP/p25 (Kd = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ42, α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.

Dynamic targeting of microtubules by TPPP/p25 affects cell survival

Recently we identified TPPP/p25 (tubulin polymerization promoting protein/p25) as a brain-specific unstructured protein that induced aberrant microtubule assemblies and ultrastructure in vitro and as a new marker for Parkinsons disease and other synucleopathies. In this paper the structural and functional consequences of TPPP/p25 are characterized to elucidate the relationship between the in vitro and the pathological phenomena. We show that at low expression levels EGFP-TPPP/p25 specifically colocalizes with the microtubule network of HeLa and NRK cells. We found that the colocalization was dynamic (tg=5 seconds by fluorescence recovery after photobleaching) and changed during the phases of mitosis. Time-lapse and immunofluorescence experiments revealed that high levels of EGFP-TPPP/p25 inhibited cell division and promoted cell death. At high expression levels or in the presence of proteosome inhibitor, green fusion protein accumulated around centrosomes forming an aggresome-like structure protruding into the nucleus or a filamentous cage of microtubules surrounding the nucleus. These structures showed high resistance to vinblastin. We propose that a potential function of TPPP/p25 is the stabilization of physiological microtubular ultrastructures, however, its upregulation may directly or indirectly initiate the formation of aberrant protein aggregates such as pathological inclusions.

Sequestration revisited: Integrating traditional electron microscopy, de novo assembly and new results

Electron microscopy analysis of the autophagic sequestration membrane (SM) in various metazoan cell types after different fixation methods shows that: (1) the growing SM cannot derive from preformed rough surfaced endoplasmic reticulum (RER) membranes by transformation; (2) the empty cleft between the two layers of the SM after aldehyde fixation is an artifact of sample preparation; (3) the SM emerges from and grows de novo in cytoplasmic areas where membranous precursors cannot be identified by traditional electron microscopy; (4) the growing SM consists of two tightly packed membrane layers with a sharp bend at the edge; (5) changes in the environment of the growing SM participate in the determination of the size and shape of the autophagosome.

Phosphorylation blocks the activity of tubulin polymerization promoting protein (TPPP): Identification of sites targeted by different kinases

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Δ3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr14, Ser18, Ser160 for Cdk5; Ser18, Ser160 for ERK2, and Ser32 for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (Kd = 2.5-2.7 μm) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr14 and/or Ser18 in its unfolded N-terminal tail.

Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils.

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.

Comparison of injection moulded, natural fibre-reinforced composites with PP and PLA as matrices

Poly(lactic acid) (PLA) and polypropylene (PP) were comparatively investigated as matrices for injection-moulded composites containing small (1–3 wt%) amounts of short sisal fibre. The morphology, thermal and dynamic mechanical properties, as well as degradation characteristics were investigated. The scanning electron microscopy (SEM) micrographs of the composites show more intimate contact and better interaction between the fibres and PLA, compared to PP. This improved interaction was confirmed by the Fourier-transform infrared (FTIR) spectroscopy results which showed the presence of hydrogen bonding interaction between PLA and the fibres. The thermal stability (as determined through thermogravimetric analysis [TGA]) of both polymers increased with increasing fibre content, with a more significant improvement in the case of PP. The differential scanning calorimetry (DSC) results showed a significant influence of the fibres on the cold crystallization and melting behaviour of PLA, even at the low fibre contents of 1–3%. The influence of the fibres on the melting characteristics of the PP was negligible, but it had a significant influence on the nonisothermal crystallization temperature range. Both the storage and loss moduli of the PLA decreased with increasing fibre content below the glass transition of PLA, but the influence on the loss modulus was more significant. The dynamic mechanical analysis (DMA) results clearly show cold crystallization of PLA around 110°C, and the presence of fibres gave rise to higher modulus values between the cold crystallization and melting of the PLA. The presence of fibres also had an influence on the dynamic mechanical properties of PP. This article further describes basic biodegradation observations for the investigated samples.

Mechanism of lysophosphatidic acid-induced amyloid fibril formation of β2-microglobulin in vitro under physiological conditions

Beta(2)-microglobulin- (beta2m-) based fibril deposition is the key symptom in dialysis-related amyloidosis. beta2m readily forms amyloid fibrils in vitro at pH 2.5. However, it is not well understood which factors promote this process in vivo, because beta2m cannot polymerize at neutral pH without additives even at elevated concentration. Here we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator, promotes amyloid formation under physiological conditions through a complex mechanism. In the presence of LPA, at and above its critical micelle concentration, native beta2m became sensitive to limited proteolytic digestion, indicating increased conformational flexibility. Isothermal titration calorimetry indicates that beta2m exhibits high affinity for LPA. Fluorescence and CD spectroscopy, as well as calorimetry, showed that LPA destabilizes the structure of monomeric beta2m inducing a partially unfolded form. This intermediate is capable of fibril extension in a nucleation-dependent manner. Our findings also indicate that the molecular organization of fibrils formed under physiological conditions differs from that of fibrils formed at pH 2.5. Fibrils grown in the presence of LPA depolymerize very slowly in the absence of LPA; moreover, LPA stabilizes the fibrils even below its critical micelle concentration. Neither the amyloidogenic nor the fibril-stabilizing effects of LPA were mimicked by its structural and functional lysophospholipid analogues, showing its selectivity. On the basis of our findings and the observed increase in blood LPA levels in dialysis patients, we suggest that the interaction of LPA with beta2m might contribute to the pathomechanism of dialysis-related amyloidosis.

Tubulin and microtubule are potential targets for brain hexokinase binding

The metabolite‐modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non‐mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme‐linked immunosorbent assay using specific anti‐brain hexokinase serum (IC50=4.0±1.4 μM). This value virtually was not affected by specific ligands such as ATP or glucose 6‐phosphate. Microtubule‐bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell‐free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.