Biochim Biophys Acta. 2015 Jan;1852(1):83-91.

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

Lehotzky A, Oláh J, Szunyogh S, Szabó A, xBerki T, Ovádi J.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest; xDepartment of Immunology and Biotechnology, Medical School, University of Pécs, Pécs, Hungary

 

ABSTRACT

Tubulin 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.

KEYWORDS: Oligodendrocyte; Tubulin Polymerization Promoting Protein/p25; Zinc

PMID: 25445539

 

SUPPLEMENT

Zinc as a ubiquitous and essential micronutrient is involved in an extraordinary range of biological functions such as growth, development and for a host of diseases, although its precise role is not fully understood. The intracellular free concentration of the bivalent zinc cation is in the nanomolar range; however, its accumulation has been detected in myelin sheath and synaptic vesicles of brain. Dyshomeostasis of zinc ions has been observed in the case of different neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s (PD) diseases [1]. The breakdown of myelin has been suggested to be associated with AD as an initiating step in the changes of the earliest stage of AD prior to the appearance of amyloid and tau pathology [2].

Tubulin Polymerization Promoting Protein (TPPP/p25), a Zn2+ binding brain-specific protein modulates the dynamics and stability of the microtubule system depending on its phosphorylation status and plays a crucial role in the differentiation of the oligodendrocytes thus the constitution of the myelin sheath [3, 4] (Scheme1).

TPPP/p25 does not have a well-defined 3D structure; it is an intrinsically disordered protein, a prototype of the Neomorphic Moonlighting Proteins, since it displays both physiological and pathological functions [5]. TPPP/p25 is crucial for the differentiation of oligodendrocytes, the major constituent of myelin sheath as well as for the formation of pathological inclusions characteristic for neurological disorders as illustrated in Scheme 1.

 

 JO FIG1

 Scheme 1 TPPP/p25 is involved in both physiological and pathological processes.

 

The protein has a zinc finger motif (His2Cys2) (His61–Cys83) within its flexible core region which is straddled by extended unstructured N- and C-terminal segments. The binding of Zn2+ to TPPP/p25 (Kd = 33 mM) induces molten globule, but not a well-defined tertiary structure formation [3]. This bivalent cation can uniquely influence its tubulin polymerization promoting and GTPase activities (cf. Scheme 2). The elevation of the intracellular Zn2+ concentration resulting in the TPPP/p25 level causing more decorated microtubule network in CHO10 and CG-4 cells expressing TPPP/p25 ectopically and endogenously, respectively.

 

JO FIG2

Scheme 2 Zinc-induced structural and functional changes.

 

The effect of zinc on the intracellular TPPP/p25 level is resulted from the Zn-induced conformational change. This effect, similarly to that produced by MG132, a proteasome inhibitor, counteracts with the proteasomal degradation of TPPP/p25. The modulating potency of zinc on the TPPP/p25 level is manifested itself in the case of either doxycycline induction or inhibition of the protein synthesis by cycloheximide. Our data suggest that the zinc as a specific divalent cation is involved in the fine-tuning of the physiological TPPP/p25 level counteracting both the enrichment and the lack of this protein leading to distinct CNS diseases (cf. Scheme 1). This is an important issue both from physiological and pathological points of view since TPPP/p25 has potency to modulate the dynamics and stability of the microtubule network by its tubulin acetylation and bundling activity [6]. The structural rearrangement of TPPP/p25 caused by zinc binding to the zinc finger motive could be a potential factor in brain physiology to optimize the intracellular TPPP/p25 level according to its physiological function.

 

References

1) B.R. Roberts, T.M. Ryan, A.I. Bush, C.L. Masters, J.A. Duce, The role of metallobiology and amyloid-beta peptides in Alzheimer’s disease, J. Neurochem. 120 (Suppl. 1) (2012) 149–166.

2) Z. Cai, M. Xiao, Oligodendrocytes and Alzheimer’s disease, Int J Neurosci. 2015, Epub ahead of print

3) A. Zotter, J. Oláh, E. Hlavanda, A. Bodor, A. Perczel, K. Szigeti, J. Fidy, J. Ovádi, Zn2+-induced rearrangement of the disordered TPPP/p25 affects its microtubule assembly and GTPase activity, Biochemistry 50 (2011) 9568–9578.

4) A. Lehotzky, P. Lau, N. Tőkési, N. Muja, L.D. Hudson, J. Ovádi, Tubulin polymerization promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation, Glia 58 (2010) 157–168.

5) J. Ovádi, Special issue Moonlighting proteins in neurological disorders, IUBMB Life (2011) 63, 453-456.

6) J. Oláh, O. Vincze, D. Virók, D. Simon, Z. Bozsó, N. Tőkési, I. Horváth, E. Hlavanda, J. Kovács, A. Magyar, M. Szűcs, F. Orosz, B. Penke, J.Ovádi, Interactions of pathological hallmark proteins: Tubulin polymerization promoting protein/p25, b-amyloid and a-synuclein. J Biol Chem. (2011) 286, 34088-34100.

 

Contact: Prof. Dr. Judit Ovádi, Institute of Enzymology, RCNS,

Hungarian Academy of Sciences

Magyar tudósok korútja 2

H-1117 Budapest, Hungary

ovadi.judit@ttk.mta.hu

http://www.enzim.hu/~ovadi/

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