Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum

Abstract

Ubiquitination is a post-translational modification implicated in a variety of cellular functions, including transcriptional regulation, protein degradation and membrane protein trafficking. Ubiquitin and the enzymes that act on it, although conserved and essential in eukaryotes, have not been well studied in parasites, despite sequencing of several parasite genomes. Several putative ubiquitin hydrolases have been identified in Plasmodium falciparum based on sequence homology alone, with no evidence of expression or function. Here we identify the first deubiquitinating enzyme in P. falciparum, PfUCH54, by its activity. We show that PfUCH54 also has deNeddylating activity, as assayed by a mammalian Nedd8-based probe. This activity is absent from mammalian homologues of PfUCH54. Given the importance of parasitic membrane protein trafficking as well as protein degradation in the virulence of this parasite, this family of enzymes may represent a target for pharmacological intervention with this disease.

Figure 1

Probe mechanisms of action. Probes were designed for the detection of enzymes capable of removing Ub and other Ub‐like proteins. An example of the Ub‐based probes is shown. Probes include a Ub moiety followed by either (A) a vinylmethylester or (B) a vinylmethylsulphone active group. Where needed, a haemagglutinin (HA) epitope tag is also included as an N‐terminal fusion. The catalytic groups act as electrophiles mimicking the C‐terminal end of the Ub gly motif and attracting DUB attack. DUBs become covalently and irreversibly attached to the probes through a thioether bond, thereby allowing for detection by anti‐HA antibodies in addition to a 10 kDa increase in size.

Figure 2

Evidence of parasite‐specific deubiquitinating and deNeddylating activity. Parasite and uninfected red blood cell (RBC) lysates were reacted with (A) HA‐Ub‐VME or (B) FLAG‐Nedd8‐VS followed by SDS‐PAGE and immunoblot with anti‐HA‐HRP or anti‐FLAG‐HRP where appropriate. Probe alone and lysate alone are shown in the left three lanes of each panel. Reaction profiles in the presence or absence of NEM are shown in the right four lanes of each panel. Numbers on the left‐hand side of (A) indicate molecular weights in kDa. The reactive polypeptide later excised for mass spectrometric analysis is indicated by the arrow. Pf, Plasmodium falciparum lysate.

Figure 3

Identification of PfUCH54 by mass spectrometry. P. falciparum and uninfected erythrocyte lysates were reacted with HA‐Ub‐VME probe on a preparative scale followed by SDS‐PAGE. Parasite‐specific reactive proteins were extracted and subjected to tandem mass spectrometry. Identified polypeptides (indicated in pink) were screened against the P. falciparum genome database and matched a 465‐amino‐acid protein annotated as a putative C‐terminal Ub hydrolase. Mass spectrometry properties of the identified peptides are shown. MH+ indicates the protonated mass of each peptide; %Mass represents the per cent of the total protein mass occupied by each peptide; AA indicates the span of amino acids covered by each peptide; and %AA identifies the per cent of amino acid coverage. Black arrows indicate potential alternative internal start sites as determined by additional translation products seen in Fig. 5.

Figure 4

Alignment of PfUCH54 with human DUB homologues and predicted structure.
A. An alignment of three human UCH‐type DUBs, UCH‐L1, UCH‐L3 and UCH37, with PfUCH54 is shown with active residues indicated by the blue arrows.
B. The crystal structure of UCH‐L3 was used to model PfUCH54. Parasite‐specific insertions are shown in green. Active site cysteine residues for both enzymes are shown in blue. The arrow indicates the trajectory of Ub that is presumed for binding to UCH‐L3.

Figure 5

Evidence of dual Ub and Nedd8 specificity for PfUCH54. In vitro transcription/translation using HA‐tagged, wild‐type PfUCH54 (left) and the catalytically inactive cysteine to serine mutant (right) as templates was performed in the presence of ³⁵S‐methionine. Translation products were reacted with the range of untagged probes followed by immunoprecipitation against HA. Analysis by autoradiography reveals Ub and Nedd8 activity specific to the wild‐type PfUCH54. Two unreactive truncated translation products are indicated by asterisk. Reactivity in the presence or absence of the alkylating agent NEM is shown for both the wild type and the mutant.