Friedeberg C et al. (2006), Identification of a structural and functional d...

XB-ART-34413

Nucleic Acids Res 2006 Jan 01;3417:4893-9. doi: 10.1093/nar/gkl434.

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Identification of a structural and functional domain in xNAP1 involved in protein-protein interactions.

Friedeberg C , Scarlett G , McGeehan J , Abu-Daya A , Guille M , Kneale G .

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xNAP1 (Xenopus nucleosome assembly protein) belongs to the family of nucleosome assembly proteins (NAPs) and shares 92% identity with human and mouse NAP1. NAPs have been reported to have a role in nucleosome assembly, cell cycle regulation, cell proliferation and transcriptional control, although the precise function of NAP1 is still not clear. Here we report the identification of a putative domain of xNAP1 by limited proteolysis. This domain has been mapped in the xNAP1 protein sequence to residues 38-282 and thus lacks the acidic sequences at the N- and C-termini. We have studied this domain and related fragments in vitro and by a functional assay involving over-expression of the protein in Xenopus laevis embryos. Analytical ultracentrifugation shows that removal of the acidic N- and C-terminal regions does not prevent the formation of larger multimers, which are predominantly hexadecamers. Injection of mRNA encoding the full-length xNAP1 or the putative domain and other related constructs into Xenopus embryos gave identical phenotypes. These results are discussed in relation to protein-protein interactions between NAP1 octamers and a possible 'squelching' mechanism.

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Species referenced: Xenopus laevis
Genes referenced: prss1 tab3 tbxt.2
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	Fig 5. Injection of any of the xNAP1 constructs leads to axial defects. (a) Injected constructs are shown with conserved regions indicated. Basic regions marked by a black box, the PEST sequence is shown in green embedded in the C-terminal basic region. The NLS is marked in red and the SFFNFF shown in purple. (b) One-cell embryos were injected with 100 pg of either full-length or truncated versions of xNAP1. Un-injected embryos from the same fertilization were cultured in parallel as controls. Embryos were grown in modified bath serum at 18 until stage 36, fixed in MEMFA (0.5 M MOPS, pH 7.4, 0.5 M EGTA, 0.5 M MgSO4 and 37% formaldehyde) and stored at −20 in methanol before photographing. All forms of injected xNAP1 gave rise to a shorter A/P axis (lack of head and tail are arrowed) than the un-injected control embryos shown in (c).
	Figure 1. Limited proteolysis of xNAP1 with trypsin and α-chymotrypsin xNAP1 (0.4 mg ml−1) was digested with either trypsin (a) or α-chymotrypsin (b) at a w/w ratio of 1:10 000 enzyme to substrate. The reaction was incubated at 25°C and stopped with 1 mM PMSF and assayed by SDS–PAGE. The most distinct fragment visible after a 2 h digestion by trypsin is ∼50 kDa (T1). A 50 kDa fragment is also visible after 2 h digestion with α-chymotrypsin (C1). (c) Further digestion for a period of 24 h with trypsin and with α-chymotrypsin reveals a fragment of ∼35 kDa in the trypsin lane (T2). A 24 h digestion with α-chymotrypsin still shows a strong fragment corresponding to C1. Two smaller fragments ∼35–37 kDa in size are labelled C2 (i) and (ii).
	Figure 2. xNAP1 amino acid sequence showing T2 and C1 N-terminal sequences. The N-terminus of the trypsin fragment T2 (red box) and the N-terminus of chymotrypsin fragments C1 (green box) are 13 amino acids apart. The predicted C-terminus of T2 and C1 are also shown (red and green, respectively). Acidic regions are underlined and the bipartite nuclear localization signal is shown in blue. Preceding the wild-type start methionine (numbered 1) is the His-tag sequence present in the recombinant T2 protein.
	Figure 3. Dynamic light scattering of the T2 domain T2 was purified by metal chelate affinity and size exclusion chromatography. Buffers contained 10% glycerol. The T2 protein sample (0.15 mg ml−1) has one major component, comprising 93.6% (w/w) of the protein with a hydrodynamic radius of 9.6 nm.
	Figure 4. Sedimentation equilibrium of the T2 domain. The sedimentation equilibrium profile of T2 (at 0.14 mg ml−1) in 10% glycerol is shown. The experiment was performed using an Optima™ XLA at 3000 r.p.m., 20°C and the scans taken at 280 nm every 5 h over 24 h. The fitted curve is for a single-species model with Mr = 430 kDa.
	Figure 5. Injection of any of the xNAP1 constructs leads to axial defects. (a) Injected constructs are shown with conserved regions indicated. Basic regions marked by a black box, the PEST sequence is shown in green embedded in the C-terminal basic region. The NLS is marked in red and the SFFNFF shown in purple. (b) One-cell embryos were injected with 100 pg of either full-length or truncated versions of xNAP1. Un-injected embryos from the same fertilization were cultured in parallel as controls. Embryos were grown in modified bath serum at 18°C until stage 36, fixed in MEMFA (0.5 M MOPS, pH 7.4, 0.5 M EGTA, 0.5 M MgSO4 and 37% formaldehyde) and stored at −20°C in methanol before photographing. All forms of injected xNAP1 gave rise to a shorter A/P axis (lack of head and tail are arrowed) than the un-injected control embryos shown in (c).

References [+] :

Abu-Daya, Zygotic nucleosome assembly protein-like 1 has a specific, non-cell autonomous role in hematopoiesis. 2005, Pubmed, Xenbase