PDBsum entry 2hdl

Cytokine

PDB id: 2hdl

Contents

Protein chain

77 a.a. *

* Residue conservation analysis

PDB id:

2hdl

Name:

Cytokine

Title:

Solution structure of brak/cxcl14

Structure:

Small inducible cytokine b14. Chain: a. Synonym: cxcl14, chemokine brak. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: cxcl14, njac, scyb14. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

F.C.Peterson,J.A.Thorpe,A.G.Harder,B.F.Volkman,S.R.Schwarze

Key ref:

F.C.Peterson et al. (2006). Structural determinants involved in the regulation of CXCL14/BRAK expression by the 26 S proteasome. J Mol Biol, 363, 813-822. PubMed id: 16987528 DOI: 10.1016/j.jmb.2006.08.057

Date:

20-Jun-06 Release date: 24-Oct-06

Protein chain

O95715  (CXL14_HUMAN) -  C-X-C motif chemokine 14 from Homo sapiens

Seq: 111 a.a.

Struc: 77 a.a.

DOI no: 10.1016/j.jmb.2006.08.057

J Mol Biol 363:813-822 (2006)

PubMed id: 16987528

Structural determinants involved in the regulation of CXCL14/BRAK expression by the 26 S proteasome.

F.C.Peterson, J.A.Thorpe, A.G.Harder, B.F.Volkman, S.R.Schwarze.

ABSTRACT

The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells; however, in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation.

Selected figure(s)

Figure 1.
Figure 1. CXCL14 expression is regulated by the 26 S proteasome and is a substrate for ubiquitylation. (a) Primary prostate epithelial cells (PrEC) and the prostate cancer cell line LNCaP were transduced for three days with CXCL14 expressing virus (pAd-CXCL14) or vector-only virus (pAd). The conditioned media were analyzed by Western blotting for CXCL14 expression. (b) Total RNA was isolated from the transduced cells, converted to cDNA and reverse transcriptase PCR performed using primers designed to detect CXCL14 or GAPDH. PCR products were separated by agarose gel electrophoresis and detected by SYBR green staining. Neg, denotes the no DNA control. (c) LNCaP cells were transduced with CXCL14 expressing adenovirus (pAd-CXCL14) for 48 h. Inhibitory doses of chloroquine (10 μM) and MG-132 (10 μM) were added for an additional 24 h. The conditioned media were analyzed by Western blotting for CXCL14 expression. Un-transduced LNCaP cells were included as a control treated with vehicle only (dimethyl sulfoxide, DMSO). (d) LNCaP cells expressing CXCL14 were treated for 24 h with increasing concentrations of lactacystin. The conditioned media were analyzed by Western blot analysis for CXCL14 expression. (e) LNCaP cells were transduced with CXCL14 expressing adenovirus (pAd-CXCL14) or vector alone. After 48 h post-transduction, cells were treated with 10 μM MG-132 or DMSO. Cells were harvested, lysed, and immunoprecipitated using the anti-CXCL14 antibody. Immune complexes were subjected to Western blot analysis and probed with a mouse anti-ubiquitin or the rabbit anti-CXCL14 antibody. A high molecular weight complex was detected which is indicative of polyubiquitylated proteins. (f) Cells were transduced with vector (pAd) or CXCL14 expressing (pAd-CXCL14) adenovirus. After 48 h post-transduction, cells were treated with MG-132 or DMSO. The media were analyzed for CXCL14 expression by Western blot analysis. Without pAd-CXCL14 transduction the protein was undetectable in all cell lines. Primary cells utilized include: prostate epithelial cells, PrEC; human urothelial cells, HUC; and kidney epithelial cells, KEC. The immortalized PrEC lines include: HPV15E6, HPVE6 oncoprotein immortalized; and HPV15E7, HPVE7 oncoprotein immortalized. Cancer cell lines include: CWR22, DU145, LNCaP, LAPC4, PC-3, PPC-1, 293 and HeLa.

Figure 4.
Figure 4. Comparison of CXCL14 and CXCL8. (a) Ribbon diagrams of CXCL14 (PDB entry 2hdl) and CXCL8 (PDB entry 1icw). Disulfide bonds are shown in yellow. (b) Overlay of CXCL14 and CXCL8 colored as in (a). Residues anchoring the N-loop to the C-terminal α-helix are shown as sticks.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2006, 363, 813-822) copyright 2006.

Figures were selected by an automated process.