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Distribution of tubulin and acetylated tubulin between soluble and polymerized fractions of treated HeLa and CG-4 cells. Cells were treated with VBL, TSA, TSA plus VBL, or paclitaxel. The soluble (sol) and polymerized (pol) tubulins were visualized by Western blot using anti-tubulin antibody and quantified by densitometry. The distribution of tubulin between the soluble (empty bar) and polymerized (black bar) fractions is illustrated by a stacked bar chart. For each sample, the total amount of tubulin was calculated by summing the values of the corresponding soluble and polymerized pools. The relative fractions were calculated by dividing the value of each pool by the total amount of tubulin. A representative result of three separate experiments is shown.
Source publication
TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differ...
Citations
... Nonetheless, these changes were observed in the presence of an almost two-fold reduction (Fu et al., 2019;Lehotzky et al., 2010;Tokési et al., 2010) in the overall density of myelinated axons at 24 (which was not certified by peer review) is the author/funder. All rights reserved. ...
... The existence of a transport system operating in mature fibres, is plausible because the myelinic channel system is developmentally derived from subcellular compartments of oligodendrocytes in which transport processes contribute directly to myelin outgrowth (Iyer et al., 2024;Nawaz et al., 2015;Snaidero et al., 2014) Analogous to neurons with growth cones, oligodendrocytes in monolayer culture have microtubules within growing cellular processes (Barry et al., 1996;Song et al., 1999) with the notable difference that these processes remain short and deliver new membranes to a flat bilayer structure that, in vivo, grows spirally around axons (Snaidero et al., 2014). Indeed, normal myelin sheath growth requires nucleation of microtubules, that is mediated by tubulin polymerization promoting protein (TPPP) (Fu et al., 2019;Lehotzky et al., 2010;Tokési et al., 2010). Further, mRNAs encoding myelin proteins and presumably the oligodendroglial translation machinery is transported on microtubules to the tip of the growing sheath (Brophy et al., 1993;Carson et al., 1998; (which was not certified by peer review) is the author/funder. ...
Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the 'myelinic channel system' contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in the myelinic channel system to maintain axonal integrity.
... In physiological conditions, TPPP modulates the dynamics and stability of the cytoskeletal microtubule system via its bundling and tubulin acetylation-promoting activities [13]. These physiological functions are mediated by its direct associations with tubulin/microtubules as well as with tubulin deacetylases such as histone deacetylase 6 and sirtuin-2 (SIRT2) [20,21]. The bundling and stabilization of microtubules result from the dimerization of the tubulinattached monomeric TPPP [22]. ...
Neurological disorders such as Parkinsonism cause serious socio-economic problems as there are, at present, only therapies that treat their symptoms. The well-established hallmark alpha-synuclein (SYN) is enriched in the inclusion bodies characteristic of Parkinsonism. We discovered a prominent partner of SYN, termed Tubulin Polymerization Promoting Protein (TPPP), which has important physiological and pathological activities such as the regulation of the microtubule network and the promotion of SYN aggregation. The role of TPPP in Parkinsonism is often neglected in research, which we here attempt to remedy. In the normal brain, SYN and TPPP are expressed endogenously in neurons and oligodendrocytes, respectively, whilst, at an early stage of Parkinsonism, soluble hetero-associations of these proteins are found in both cell types. The cell-to-cell transmission of these proteins, which is central to disease progression, provides a unique situation for specific drug targeting. Different strategies for intervention and for the discovery of biomarkers include (i) interface targeting of the SYN-TPPP hetero-complex; (ii) proteolytic degradation of SYN and/or TPPP using the PROTAC technology; and (iii) depletion of the proteins by miRNA technology. We also discuss the potential roles of SYN and TPPP in the phenotype stabilization of neurons and oligodendrocytes.
... Predominantly found in structures containing long-lived stable microtubules (cilia, flagella and centrioles), abnormal acetylation has also been reported in neurodegenerative disorders. In Parkinson's, for example, aberrant microtubule structures such as perinuclear cages are formed, mediated by tubulin polymerisation-promoting protein/p25 (TPPP/p25) [54]. We therefore attribute our findings to the maintained presence of IL-13 in culture media, inducing cellular stress and consequentially, aberrant acetylation of tubulin. ...
Equine asthma, previously known as Recurrent Airway Obstruction (RAO) or Inflammatory Airway Disease (IAD), is an often-debilitating condition that may severely affect both performance and quality of life. Research is hindered by the low sample numbers of subjects recruited to studies, a consequence in part of the invasive nature of the sampling methods of bronchial brushing and biopsy. We present an alternative method of sampling equine airway epithelial cells, the ‘nasal brush method’ (NBM). Obtained by light brushing of the ventral meatus whilst the horse is under standing sedation, these cells express the same markers of differentiation as their deeper counterparts. Grown as 3-D spheroids or as air-liquid interface cultures, nasal epithelial cells are responsive to the inflammatory cytokine interleukin-13. This may be attenuated by modulation of the Notch signalling pathway using the gamma-secretase inhibitor Semagecestat; a previously unreported finding that cements the link between equine and human asthma research and strengthens the case for a One Health approach in researching asthma pathophysiology and therapeutic intervention.
... by HDAC6 inhibition, it is not clear that microtubule function is responsible for inflammation in CF since HDAC6 does have other targets [17][18][19] . Tubulin polymerization-promoting protein (Tppp) is a regulator of microtubule polymerization and promotes acetylation of alpha tubulin by binding to and inhibiting HDAC6 20 . We have previously shown that knocking down expression of Tppp in a human tracheal epithelial cell line increases inflammatory signaling consistent with what is seen in CF cells 12 . ...
... Previous work of ours demonstrates distinct changes to microtubule regulation in CF cells consisting of reduced tubulin acetylation and slower rates of microtubule reformation 8,9 . We have also demonstrated that knocking down expression of Tppp in an immortalized epithelial cell line reproduced these microtubule phenotoypes, consistent with the function of Tppp as an enhancer of microtubule elongation and as an HDAC6 inhibitor 12,20 . Inhibition of HDAC6 has been shown to have significant benefit in addressing multiple CF phenotypes 5,21,26 . ...
Microtubule dysfunction has been implicated as a mediator of inflammation in multiple diseases such as disorders of the cardiovascular and neurologic systems. Tubulin polymerization promoting protein (Tppp) facilitates microtubule elongation and regulates tubulin acetylation through inhibition of cytosolic deacetylase enzymes. Pathologic alterations in microtubule structure and dynamics have been described in cystic fibrosis (CF) and associated with inflammation, however the causality and mechanism remain unclear. Likewise, Tppp has been identified as a potential modifier of CF airway disease severity. Here we directly assess the impact of microtubule dysfunction on infection and inflammation by interrogating wild type and a Tppp knockout mouse model (Tppp − / −). Mice are challenged with a clinical isolate of Pseudomonas aeruginosa-laden agarose beads and assessed for bacterial clearance and inflammatory markers. Tppp − / − mouse model demonstrate impaired bacterial clearance and an elevated inflammatory response compared to control mice. These data are consistent with the hypothesis microtubule dysregulation is sufficient to lead to CF-like airway responses in mice.
... a TPPP-derived differentiated OLG proceeds to aggregation by SYN leading to the formation of intracellular inclusion found in multiple system atrophy33,74 . b TPPP and/or SYN-induced aggregation of SYN in neuron; SYN-TPPP assembly-promoted Lewy body characteristic of Parkinson's disease75 . Atomic-resolution structure of alpha-synuclein fibrils (PDB ID: 2N0A)76 .V. Norris et al. ...
Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are central to many physiological functions and to various pathologies that include neurodegeneration. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs that we term Phenotype-Preserving Disordered Proteins (PPDP) play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.
... Specifically, in the study of Dilworth et al [23], the majority of tubulin was in the soluble nonpolymerized fraction from U2OS cells, while we found most of the tubulin was in the insoluble polymerized fraction in C2C12 cells (Fig. 4A). Interestingly, the relative proportion of soluble to insoluble tubulin that we found in C2C12 myoblasts is similar to that reported for HELA, CG-4 cells and OE19 cell lines [43,44]. The reason for these cell type differences is not clear, however, they suggest the possibility of cell-specific differences in MT dynamics. ...
FKBP25 (FKBP3 gene) is a dual‐domain PPIase protein that consists of a C‐terminal PPIase domain and an N‐terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role of FKBP25 in non‐transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline‐inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.
... It binds to MTs and lowers the growth rate of 'plus' end, thus protecting the MTs from phases of depolymerization. TPPP/p25 increases MT acetylation via putative inhibition of HDAC6 [91]. It is generally overexpressed in oligodendroglial cells (OLGs) in the normal brain, but during synucleinopathies such as PD and multiple system atrophy, colocalization and abundant coexpression of α-syn and TPPP/ p25 are observed in Lewy bodies and cytoplasmic glial cells. ...
The inter-neuronal communication occurring in extensively branched neuronal cells is achieved primarily through the microtubule (MT)-mediated axonal transport system. This mechanistically regulated system delivers cargos (proteins, mRNAs and organelles such as mitochondria) back and forth from the soma to the synapse. Motor proteins like kinesins and dynein mechanistically regulate polarized anterograde (from the soma to the synapse) and retrograde (from the synapse to the soma) commute of the cargos, respectively. Proficient axonal transport of such cargos is achieved by altering the microtubule stability via post-translational modifications (PTMs) of α- and β-tubulin heterodimers, core components constructing the MTs. Occurring within the lumen of MTs, K40 acetylation of α-tubulin via α-tubulin acetyl transferase and its subsequent deacetylation by HDAC6 and SIRT2 are widely scrutinized PTMs that make the MTs highly flexible, which in turn promotes their lifespan. The movement of various motor proteins, including kinesin-1 (responsible for axonal mitochondrial commute), is enhanced by this PTM, and dyshomeostasis of neuronal MT acetylation has been observed in a variety of neurodegenerative conditions, including Alzheimer’s disease and Parkinson’s disease (PD). PD is the second most common neurodegenerative condition and is closely associated with impaired MT dynamics and deregulated tubulin acetylation levels. Although the relationship between status of MT acetylation and progression of PD pathogenesis has become a chicken-and-egg question, our review aims to provide insights into the MT-mediated axonal commute of mitochondria and dyshomeostasis of MT acetylation in PD. The enzymatic regulators of MT acetylation along with their synthetic modulators have also been briefly explored. Moving towards a tubulin-based therapy that enhances MT acetylation could serve as a disease-modifying treatment in neurological conditions that lack it.
Graphical abstract
... a: TPPP-derived differentiated OLG proceeds to aggregation by SYN leading to the formation of intracellular inclusion found in multiple system atrophy [34,69]. b: TPPP and/or SYN-induced aggregation of SYN in neuron; SYN-TPPP assembly-promoted Lewy body characteristic of Parkinson's disease [70]. Atomic-resolution structure of alpha-synuclein brils (PDB ID: 2N0A) [71]. ...
Intrinsically disordered proteins (IDPs) are central to many physiological functions and to various pathologies that include neurodegeneration. They can interact with numerous partner proteins. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs, which we term Phenotype-Preserving Disordered Proteins (PPDP), play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.
... 3C). Tppp is a multiple effector of the microtubule organization that not only protects the microtubules from cold depolymerization via its microtubule bundling activity, but also increases the level of acetylation by inhibiting the activity of histone deacetylase 6 (Tőkési et al. 2010), rendering microtubules more mechanically stable ). As the rapidly evolving genes and positively selected genes identified in the Amur sleeper are known to be important for cytoskeletal stability, especially in tubulin biogenesis, this suggests that these genes have been co-opted to prevent irreparable structural damage to the cytoskeletal system and maintain normal function over the freeze/thaw cycle. ...
Freeze tolerance, the ability of an organism to survive internal ice formation, is a striking survival strategy employed by some ectotherms living in cold environments. However, the genetic bases of this remarkable adaptation are largely unknown. The Amur sleeper (Perccottus glenii), the only known freeze-tolerant fish species, can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate its strategies for surviving freezing. Evolutionary analysis suggested that the Amur sleeper diverged from its closest non-cold-hardy relative about 15.07 million years ago and has experienced a high rate of protein evolution. Transcriptomic and metabolomic data identified a coordinated and tissue-specific regulation of genes and metabolites involved in hypometabolism, cellular stress response, and cryoprotectant accumulation involved in freezing and thawing. Several genes show evidence of accelerated protein sequence evolution or family size expansion were found as adaptive responses to freezing induced stresses. Specifically, genetic changes associated with cytoskeleton stability, cryoprotectant synthesis, transmembrane transport and neuroprotective adaptations were identified as potentially key innovations that aid in freezing survival. Our work provides valuable resources and opportunities to unveil the molecular adaptations supporting freeze tolerance in ectothermic vertebrates.
... The pBiFC-V N -TPPP1 and pBiFC-V C -SYN were as previously described [33,42]. Prokaryotic expression vector containing the insert for human TPPP1 and TPPP3 were prepared and purified as previously described [10,43]. Structures and sequences of all constructs were verified by restriction mapping and DNA sequencing. ...
... Previously, we showed that TPPP1 regulates the dynamics and stability of the microtubule network through its acetylation-enhancing activity by inhibiting the cytosolic tubulin deacetylase enzymes, histone deacetylase 6 and sirtuin-2 [43,55]. We tested the effect of TPPP3, as compared to that of TPPP1, on the microtubule acetylation by Western blot in HeLa cells expressing TPPP3 and TPPP1, as described in the Materials and Methods Section. ...
Parkinson’s disease is characterized by locomotion deficits, dopaminergic neuronal loss and alpha-synuclein (SYN) aggregates; the Tubulin Polymerization Promoting Protein (TPPP/p25 or TPPP1) is also implicated in these processes. The moonlighting and chameleon TPPP1 modulates the dynamics/stability of the multifunctional microtubule network by promoting its acetylation and bundling. Previously, we identified the microtubule-associated TPPP3, a homologue of TPPP1 lacking its N-terminus; however, its involvement in physiological or pathological processes was not elucidated. In this work, we have shown the modulatory role of TPPP3, similarly to TPPP1, in microtubule organization, as well as its homo- and hetero-associations with TPPP1. TPPP3, in contrast to TPPP1, virtually does not bind to SYN; consequently, it does not promote SYN aggregation. Its anti-aggregative potency is achieved by counteracting the formation of the TPPP1–SYN pathological complex/aggregation leading to Parkinsonism. The interactions of TPPP3 have been determined and quantified in vitro with recombinant human proteins, cell extracts and in living human cells using different methods including bifunctional fluorescence complementation. The tight association of TPPP3 with TPPP1, but not with SYN, may ensure a unique mechanism for its inhibitory effect. TPPP3 or its selected fragments may become a leading agent for developing anti-Parkinson agents.
