ADP-ribosylation Factor Family Of GTP-binding Proteins Research Articles

Structure of an ADP-ribosylation factor, ARF1, from Entamoeba histolytica bound to Mg(2+)-GDP.

Entamoeba histolytica is the etiological agent of amebiasis, a diarrheal disease which causes amoebic liver abscesses and amoebic colitis. Approximately 50 million people are infected worldwide with E. histolytica. With only 10% of infected people developing symptomatic amebiasis, there are still an estimated 100,000 deaths each year. Because of the emergence of resistant strains of the parasite, it is necessary to find a treatment which would be a proper response to this challenge. ADP-ribosylation factor (ARF) is a member of the ARF family of GTP-binding proteins. These proteins are ubiquitous in eukaryotic cells; they generally associate with cell membranes and regulate vesicular traffic and intracellular signalling. The crystal structure of ARF1 from E. histolytica has been determined bound to magnesium and GDP at 1.8 Å resolution. Comparison with other structures of eukaryotic ARF proteins shows a highly conserved structure and supports the interswitch toggle mechanism of communicating the conformational state to partner proteins.

36] Purification and characterization of GIT family of ADP-ribosylation factor (ARF) GTPase-activating proteins

This chapter describes the purification of recombinant GIT1 and GIT2, and the assay of these proteins as ARF GAPs. The ARF family of GTP-binding proteins consists of six distinct ARF proteins, as well as several ARF-like (ARL) proteins and the ARF domain 1 (ARD1) protein. Individual recombinant ARF proteins are expressed in E. coli using a T7 RNA polymerase system and purified using ion exchange chromatography on DEAE-Sephacel and gel filtration chromatography using UltraGel AcA 54. The ability of GAPs to alter the guanine nucleotides bound to ARF proteins is assessed using a single-round GTP hydrolysis assay. ARF proteins are incubated with [α- 32 p]GTP to create the substrate for the GAP proteins (GTP-bound ARF) prior to addition of any GAP proteins.

ADP-ribosylation factor (ARF)-like 3, a new member of the ARF family of GTP-binding proteins cloned from human and rat tissues.

The human and rat homologues of a new member of the ADP-ribosylation factor (ARF) family of 21-kDa GTP-binding proteins, termed Arl3, were identified as an expressed sequence tag (human) and as a product of polymerase chain reaction amplification using degenerate probes derived from conserved sequences in members of the ARF family (rat). Alignments of the full-length open reading frames of the human and rat homologues revealed the encoded proteins to be over 97% identical to each other and 43% identical to human ARF1. Northern blots of mRNA from seven human tissues and four rat tissues revealed the presence of a ubiquitous band of about 1 kilobase in length that hybridized with the corresponding Arl3 probes. A number of human tumor cell lines expressed Arl3, as determined by immunoblotting with an Arl-specific antibody, raised against a peptide derived from the human Arl3 sequence. The level of Arl3 expressed in these cell lines was on the order of 0.01% of total cell protein. Purified recombinant human Arl3 was shown to bind guanine nucleotides but lacks ARF activity and intrinsic or ARF GTPase-activating protein-stimulated GTPase activity. In contrast to ARF proteins, the Arl3 protein has reduced dependence on phospholipids and magnesium for guanine nucleotide exchange. Thus, Arl3 is a ubiquitously expressed GTP-binding protein in the ARF family with distinctive biochemical properties consistent with its having unique, but unknown, role(s) in cell physiology.