Investigation of the SecinH3/tubulin interaction

Table 5.6 – Continued

Experimental Calculated

Name m/z z m m ∆m Score Sequence

TBA1C HUMAN 1015.5 1 1014.5 1014.6 -0.1 11 DVNAAIATIK Tubulin 543.5 2 1085.0 1084.6 0.4 50 EIIDLVLDR alpha-1C chain 625.4 2 1248.8 1248.5 0.2 50 YMACCLLYR Mass: 50548 800.0 2 1598.0 1597.8 0.2 31 TIQFVDWCPTGFK Score: 244 851.6 2 1701.2 1700.9 0.3 46 AVFVDLEPTVIDEVR

573.7 3 1718.1 1717.9 0.2 17 NLDIERPTYTNLNR 879.1 2 1756.2 1756.0 0.2 47 IHFPLATYAPVISAEK 913.2 2 1824.4 1824.0 0.4 38 VGINYQPPTVVPGGDLAK 1004.5 2 2007.0 2006.9 0.1 88 TIGGGDDSFNTFFSETGAGK

922.2 3 2763.6 2763.3 0.3 8 AYHEQLTVAEITNACFEPA-NQMVK

Surprisingly, ARNO was not identified. Actually, even in absence of proteome it was difficult to detect the ∆PBR signal. In fact, only in one of two experiments it was possible to unambiguously identify one ∆PBR peptide in the enriched sample (Table 5.5). Since the signal intensity was already particularly low, it was improbable to find ARNO signals in the proteome sample, where other (biotinylated) proteins are competing.

Furthermore, in the same experiment, an unexpected observation was made. In fact, labelling of proteome with SecinH3-TPD resulted in identification of various tubulin isotypes. For example, more than 10 peptide matches with good scores were found for “Tubulin beta chain” (Table 5.6). With a sequence coverage of 35 %, this hit can definitely be trusted. Thus, I decided to investigate the interaction of SecinH3-TPD with tubulin in more detail.

Figure 5.31: Microtubules in the cell -a. An in-terphase cell stained with an antibody to tubulin. Mi-crotubules extend from the centrosome throughout the cell. b. A schematic diagram of the cell. Centrioles are shown in the centrosome (yellow). Red circles denote vesicles moving to the outside of the cell. Green circles denote vesicles moving to the centrosome.¹³⁷Reprinted by permission from Macmillan Publishers Ltd: Nature (Ref. 137), copyright 2003.

5.3.7.1 Tubulin and the microtubules

Tubulin is the heterodimeric building block of microtubules and, as such, involved in various essential cell functions. Microtubules, as part of the cytoskeleton, offer mechanical support for the cell shape and provide tracks for transport of vesicle and organelles (Fig. 5.31). Moreover, the mitotic spindle they form during cell division is required for the correct segregation of chromosomes.

These various functions are highly regulated at different levels. Additionally to the transcription of different tubulin isotypes, post-translational modifications (PTMs) and interaction with microtubule-associated proteins (MAPs) further tune the nucleotide regulated microtubule dynamics¹³⁸.

Microtubules dynamic instability

By head-to-tail association of theα-β tubulin dimers, linear protofilaments are formed, which, in turn, lead to the formation of the cylindric microtubules by lateral association.

The polarity of the dimer is maintained during the polymerisation process and mirrored in the microtubules polarity¹³⁷ (Fig. 5.32a). In animal cells, the minus end, which is terminated by the α subunit, is generally anchored at centrosomes and the plus

end grows toward the periphery, resulting in the characteristic radiating pattern^{137, 139} (Fig. 5.31).

Figure 5.32: Microtubule structure and dynamics. -a. A microtubule lattice. The beta-subunit of tubulin is on the plus end. b. Dynamic instability of microtubules. Micro-tubules growing out from a centrosome switch between phases of growing and shrinking.

The figure shows a hypothetical aster at two different times. The different colours represent different microtubules. The red and yellow microtubules are shrinking at both times. The blue microtubule is growing at both times. The green microtubule, growing at the first time, has undergone a catastrophe by the second time. The brown microtubule, shrinking at the first time, has undergone a rescue by the second time.¹³⁷Reprinted by permission from Macmillan Publishers Ltd: Nature (Ref. 137), copyright 2003.

Microtubules polymerisation is a highly dynamic process. In fact, microtubules are subject to stochastic switch between growing and shrinking phases (Fig. 5.32b). This property, called dynamic instability¹⁴⁰, is essential to their function and depend on the GTPase activity of tubulin¹⁴¹.

Indeed, each heterodimer subunit carries a guanine nucleotide. While GTP in the α unit is buried at the monomer-monomer interface within the dimer, and is thus non-exchangeable (N-site), inβtubulin it sits on the dimer surface and is fully exchangeable (E-site)¹⁴². For polymerisation to occur, the E-site has to be GTP-loaded, but, at the same time, binding of a new dimer triggers hydrolysis in the adjacent E-site. As a result, the microtubule body is composed of GDP-tubulin subunits and is in an energetically unfavorable state¹⁴¹ which favours shrinking events (Fig. 5.33b). In fact, GDP-tubulin is thought to have a bent conformation which places stress on the microtubules lattice.

During depolimerisation the ends curls, releasing the strain^{137, 141} (Fig. 5.33d).

An additional level of regulation is offered by MAPs. By interacting with the solu-ble, non-polymerized tubulin subunits, the microtubule wall lattice and/or microtubule

Figure 5.33: Model for how the GTP hydrolysis cycle is coupled to structural changes in the microtubule. - a. Atomic structure of the tubulin dimer as seen in the wall of the protofilament. b. Docking of the alpha-beta subunit to the microtubule end. Residues from the incoming alpha-subunit trigger hydrolysis of the GTP bound to the lattice-attached beta-subunit. c, d. Microtubules at growing ends contain sheets of protofilaments while microtubules at shrinking ends curl. The straight-bent transition is also shown in panel d. The GTP dimer is thought to have a straight conformation that fits nicely into the straight wall of the microtubule. Hydrolysis of GTP induces a bend in the subunit, but this bend is constrained within the lattice. The constraint places stress on the lattice, which is released during depolymerization, allowing the protofilament to adopt a curled conformation.¹³⁷ Reprinted by permission from Macmillan Publishers Ltd: Nature (Ref. 137), copyright 2003.

ends, they regulate microtubule stability and dynamics (See Refs. 143 and 144 for re-view).

When it comes to assigning a specific function to a microtubule, PTMs play a prominent role. In fact, a considerable number of reversible PTMs, such as acetylation, polyglycylation, polyglutamylation, tyrosination/detyrosination, phosphorylation, and palmitoylation contribute to microtubules diversity¹⁴⁵. Most PTMs occur on micro-tubules rather than on unpolymerized tubulin and stable micromicro-tubules accumulate more modifications than dynamic microtubules¹⁴⁶. Since differences in PTM patterns can be seen between stable microtubules, the PTMs are postulated to play a role in their specific functions (see Refs. 145 and 146 for review).

Microtubule-targeting drugs

Since during mitosis highly coordinated microtubule dynamics is required, compounds which interfere with microtubule dynamics limits proliferation and have been used as anticancer drugs for years. They are classified as microtubule stabilizers or destabilizers

but both classes inhibit mitosis through a similar mechanism of slowing microtubule dynamics, resulting in mitotic arrest and apoptosis¹⁴⁷.

An additional classification is done by the binding site on tubulin. Indeed, desta-bilising drugs are usually binding to the vinca- and colchicine-sites, while microtubule stabilisers mostly bind the taxane-site (but inhibitors binding at other sites were re-cently discovered). For a detailed explanation of the mechanisms of action, please read Ref. 147.

5.3.7.2 Labelling of purified tubulin

Figure 5.34: SecinH3-TPD binds∆PBR preferentially over tubulin- Labelling of purified tubulin was tested in the standard labelling assay (1µM protein, 20µM SecinH3-TPD in 10 % DMSO). After separation on a 7.5 % SDS-PAGE and Western blotting, the labelled proteins were detected with a NeutrAvidin DyLight 800 fluorescent conjugate. The total amount of protein was detected by Coomassie staining of gel. The strong tagging of tubulin is competed by ∆PBR.

Because of the essential role of tubulin in cells, it was particularly important to investigate if SecinH3 is specifically binding to tubulin and interfering with its functions.

As a first control, purified tubulin was analysed in the standard labelling assay. As evident in Figure 5.34, SecinH3-TPD strongly labelled tubulin, but the binding was competed by ∆PBR. Indeed, while labelling of tubulin was drastically decreased in the presence of ∆PBR, ARNO signal remained stable. This result shows that SecinH3-TPD interacts with tubulin but,in vitro, SecinH3-TPD preferentially binds ARNO.

5.3.7.3 Competition with SecinH3

To exclude that SecinH3-TPD interaction with tubulin is mediated by the photoreactive or desthiobiotin moieties, a competition experiment was performed. Labelling was carried out in the presence of non derivatised SecinH3 in excess. The clear inhibition of

Figure 5.35: SecinH3 competes labelling of both tubulin and ∆PBR - Non derivatised SecinH3 was added in excess to labelling reactions with 2.5µM SecinH3-TPD.

After separation on a 7.5 % SDS-PAGE and Western blotting, the labelled proteins were detected with a NeutrAvidin DyLight 800 fluorescent conjugate. Tagging of both tubulin and ∆PBR was significantly reduced by the competitor.

labelling visible in Figure 5.35 in the competed sample, accords binding to the SecinH3 core.

5.3.7.4 Analysis of SecinH3 effect on microtubules structure

The most difficult question to answer was if the SecinH3-tubulin interaction is of bi-ological relevance. To analyse a possible effect of SecinH3 on microtubule structure, immunofluorescence was used. H460 cells were treated overnight with SecinH3 or the microtubule-targeting drugs demecolcine and paclitaxel prior fixation in methanol. Mi-crotubules were visualized by immunofluorescence using a FITC conjugated monoclonal anti-α-tubulin antibody.

In Figure 5.36, two representative views for each condition are shown. It is read-ily visible that mitosis is heavread-ily impaired in the demecolcine and paclitaxel treated cells (these drugs binds the colchicine- and taxane-site, respectively). Additionally, in the lower paclitaxel panel, bundling of interphase microtubules, a typical effect of taxane^{147, 148}, is apparent. In contrast, in SecinH3 treated cells the mitotic spindle is still formed (upper panel) and no grave defect is visible in the interphase cells. This results exclude a major impact of SecinH3 on microtubules dynamic. Possible smaller

effects, for example on PTMs or interaction with MAPs, can not be ruled out.

Figure 5.36: SecinH3 has no evident effect on microtubules structure - H460 cells were treated with SecinH3, demecolcine or paclitaxel, fixed in metanol and the micro-tubules structure visualized with a FITC conjugated monoclonal anti-α-tubulin antibody by immunofluorescence. In the demecolcine and paclitaxel treated cells, impairment of mitotic spindle formation is evident. The tipical bundling of interphase microtubules is evident for paclitaxel treated cells (lower panel). In contrast, no evident defect is visible in SecinH3 treated cells and the mitotic spindle is still formed (upper panel).