Fibronectin splice variants: Understanding their multiple roles in health and disease using engineered mouse models

The extracellular matrix (ECM) is a highly dynamic network of proteins, glycoproteins, and proteoglycans. Numerous diseases result from mutation in genes coding for ECM proteins, but only recently it has been reported that mutations in the fibronectin (FN) gene were associated with a human disorder. FN is one of the main components of the ECM. It generates protein diversity through alternative splicing of a single pre‐mRNA, having at least 20 different isoforms in humans. The precise function of these protein isoforms has remained obscure in most cases. Only in the recent few years, it was possible to shed light on the multiple roles of the alternatively spliced FN isoforms. This substantial progress was achieved basically with the knowledge derived from engineered mouse models bearing subtle mutations in specific FN domains. These data, together with a recent report associating mutations in the FN gene to a form of glomerulopathy, clearly show that mutations in constitutive exons or misregulation of alternatively spliced domains of the FN gene may have nonlethal pathological consequences. In this review, we focus on the pathological consequences of mutations in the FN gene, by connecting the function of alternatively spliced isoforms of fibronectin to human diseases. © 2011 IUBMB IUBMB Life, 63(7): 538–546, 2011Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86988/1/493_ftp.pd

Dual life of TPPP/p25 evolved in physiological and pathological conditions

Neomorphic moonlighting proteins perform distinct functions under physiological and pathological conditions without alterations at the gene level. The disordered tubulin-polymerization-promoting protein (TPPP/p25), a prototype of neomorphic moonlighting proteins, modulates the dynamics and stability of the microtubule system via its bundling and tubulin acetylation-promoting activities. These physiological functions are mediated by its direct associations with tubulin/microtubules as well as tubulin deacetylases such as histone deacetylase (HDAC) 6. In a normal brain, TPPP/p25 is expressed in oligodendrocytes and plays a crucial role in the formation of projections in the course of differentiation required for axon ensheathment. Under pathological conditions, TPPP/p25 interacts with α-synuclein, an aberrant protein–protein interaction resulting in aggregation leading to the formation of inclusions as clinical symptoms. The co-enrichment and co-localization of TPPP/p25 and α-synuclein were established in human-brain inclusions characteristic of Parkinson's disease (PD) and other synucleinopathies. The binding segments on TPPP/p25 involved in the physiological and the pathological interactions were identified and validated at molecular and cellular levels using recombinant proteins and transfected HeLa and inducible Chinese-hamster ovary (CHO) 10 cells expressing TPPP/p25. Our finding that distinct motifs are responsible for the neomorphic moonlighting feature of TPPP/p25, has powerful innovative effects in anti-Parkinson's disease drug research.</jats:p

Sensor potency of the moonlighting enzyme-decorated cytoskeleton

Background: There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis: In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis: Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis: The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined

Targeting the interface of the pathological complex of α-synuclein and TPPP/p25

AbstractThe pathological interaction of intrinsically disordered proteins, such as α-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25), is often associated with neurodegenerative disorders. These hallmark proteins are co-enriched and co-localized in brain inclusions of Parkinson's disease and other synucleinopathies; yet, their successful targeting does not provide adequate effect due to their multiple functions. Here we characterized the interactions of the human recombinant wild type SYN, its truncated forms (SYN1–120, SYN95–140), a synthetized peptide (SYN126–140) and a proteolytic fragment (SYN103–140) with TPPP/p25 to identify the SYN segment involved in the interaction. The binding of SYN103–140 to TPPP/p25 detected by ELISA suggested the involvement of a segment within the C-terminal of SYN. The studies performed with ELISA, Microscale Thermophoresis and affinity chromatography proved that SYN95–140 and SYN126–140 – in contrast to SYN1–120 – displayed significant binding to TPPP/p25. Fluorescence assay with ANS, a molten globule indicator, showed that SYN, but not SYN1–120 abolished the zinc-induced local folding of both the full length as well as the N- and C-terminal-free (core) TPPP/p25; SYN95–140 and SYN126–140 were effective as well. The aggregation-prone properties of the SYN species with full length or core TPPP/p25 visualized by immunofluorescent microscopy demonstrated that SYN95–140 and SYN126–140, but not SYN1–120, induced co-enrichment and massive intracellular aggregation after their premixing and uptake from the medium. These data with their innovative impact could contribute to the development of anti-Parkinson drugs with unique specificity by targeting the interface of the pathological TPPP/p25-SYN complex

Microtubule-Associated Proteins with Regulatory Functions by Day and Pathological Potency at Night

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeleton, including the microtubule network. Microtubules play crucial roles achieved by their decoration with proteins/enzymes as well as by posttranslational modifications. This review focuses on the Tubulin Polymerization Promoting Protein (TPPP/p25), a new microtubule associated protein, on its "regulatory functions by day and pathological functions at night". Physiologically, the moonlighting TPPP/p25 modulates the dynamics and stability of the microtubule network by bundling microtubules and enhancing the tubulin acetylation due to the inhibition of tubulin deacetylases. The optimal endogenous TPPP/p25 level is crucial for its physiological functions, to the differentiation of oligodendrocytes, which are the major constituents of the myelin sheath. Pathologically, TPPP/p25 forms toxic oligomers/aggregates with α-synuclein in neurons and oligodendrocytes in Parkinson's disease and Multiple System Atrophy, respectively; and their complex is a potential therapeutic drug target. TPPP/p25-derived microtubule hyperacetylation counteracts uncontrolled cell division. All these issues reveal the anti-mitotic and α-synuclein aggregation-promoting potency of TPPP/p25, consistent with the finding that Parkinson's disease patients have reduced risk for certain cancers

Zinc-induced structural changes of the disordered TPPP/p25 inhibits its degradation by the proteasome

AbstractTubulin Polymerization Promoting Protein/p25 (TPPP/p25), a neomorphic moonlighting protein displaying both physiological and pathological functions, plays a crucial role in the differentiation of the zinc-rich oligodendrocytes, the major constituent of myelin sheath; and it is enriched and co-localizes with α-synuclein in brain inclusions hallmarking Parkinson's disease and other synucleinopathies. In this work we showed that the binding of Zn2+ to TPPP/p25 promotes its dimerization resulting in increased tubulin polymerization promoting activity. We also demonstrated that the Zn2+ increases the intracellular TPPP/p25 level resulting in a more decorated microtubule network in CHO10 and CG-4 cells expressing TPPP/p25 ectopically and endogenously, respectively. This stabilization effect is crucial for the differentiation and aggresome formation under physiological and pathological conditions, respectively. The Zn2+-mediated effect was similar to that produced by treatment of the cells with MG132, a proteasome inhibitor or Zn2+ plus MG132 as quantified by cellular ELISA. The enhancing effect of zinc ion on the level of TPPP/p25 was independent of the expression level of the protein produced by doxycycline induction at different levels or inhibition of the protein synthesis by cycloheximide. Thus, we suggest that the zinc as a specific divalent cation could be involved in the fine-tuning of the physiological TPPP/p25 level counteracting both the enrichment and the lack of this protein leading to distinct central nervous system diseases

Pharmacological targeting of α-synuclein and TPPP/p25 in Parkinson's disease

With the aging of population, neurological disorders, and especially disorders involving defects in protein conformation (also known as proteopathies) pose a serious socio-economic problem. So far there is no effective treatment for most proteopathies, including Parkinson's disease (PD). The mechanism underlying PD pathogenesis is largely unknown, and the hallmark proteins, α-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25) are challenging drug targets. These proteins are intrinsically disordered with high conformational plasticity, and have diverse physiological and pathological functions. In the healthy brain, SYN and TPPP/p25 occur in neurons and oligodendrocytes, respectively; however, in PD and multiple system atrophy, they are co-enriched and co-localized in both cell types, thereby marking pathogenesis. Although large inclusions appear at a late disease stage, small, soluble assemblies of SYN promoted by TPPP/p25 are pathogenic. In the light of these issues, we propose a new innovative strategy for the validation of a specific drug target based upon the identification of contact surfaces of the pathological SYN-TPPP/p25 complex that may lead to the development of peptidomimetic foldamers suitable for pharmaceutical intervention. This article is protected by copyright. All rights reserved

TPPP/p25 promotes tubulin acetylation by inhibiting histone deacetylase 6.

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 differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of α-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of α-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 μm concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization