Cytohesin

FIGURE 64 Virtual screening for cytohesin inhibitors, (a) VS workflow. Detailed description of the workflow leading to selected candidate compounds and experimentally confirmed hits, (b) Structures of reference compound and preferred hits. The reference compound SecinHS and the best hits from each of two VS rounds, Secinl6 and SecinBT, respectively, are shown together with their carbon skeletons. The potency of each compound is also reported. For color details, please see color plate section. 

FIGURE 6A Virtual screening for cytohesin inhibitors, (a) VS workflow. Detailed description of the workflow leading to selected candidate compounds and experimentally confirmed hits. 

FIGURE 6.4 (c) NSGs capturing different stages of the cytohesin inhibitor VS projects. 

Structure and Function of Cytohesin-1 Organization of Cytohesin Genes... 

PSCD4 (cytohesin-4) located, respectively on chromosomes 17q25 (Dixon et al., 1993), 19ql3.3 (Buchet-Poyau et al., 2002), 7p22.1 (Kim, 1998) and 22ql2.3-ql3.1 (Ogasawara et ah, 2000). Overall, the four human cytohesin mRNAs are 80% identical. 

The cytohesin-1 molecule (diagram in Fig. 1 see for review [Moss and Vaughan, 2002]) is 77% identical in amino acid sequence to cytohesin-4 and 90% identical to the other two cytohesins. All four human cytohesins contain at the N-terminus a coiled-coil domain of a 60 amino acids, a centrally located Sec7 domain of 200 residues, and a PH domain of 100 residues. The greatest variability among cytohesins is found in the C-terminal 30 amino acids. 

In addition to activating ARF proteins, cytohesin-1, but not cytohesin-2, activated the ADP-ribosylation factor-domain protein 1 ARDl... 

Fig. 2. Effect of cytohesin-1 and C-1 Sec7 on [ S] GTP7S binding by hARFl. Both cytohesin -1 (A) and its Sec7 domain ( ) accelerate the binding of GTP7S in a concentration dependent manner (reprinted with permission from Pacheco-Rodriguez et al., 1998).

Pacheco-Rodriguez et al, 1998 Vitale et al., 2000). Interaction with the ARF domain of ARDl required lysine 91 in the cytohesin-1 See domain (Vitale et ai, 2000), which is not within the sequences (motifs 1 and 2) that are critical for GEP activity. The 64-kDa ARD-1 was discovered and cloned because of its C-terminal 18-kDa ARF domain (Mishima et al., 1993). Its GTPase-activating domain was later recognized (Vitale et al., 1996). The molecular structure of ARDl identified it as a member of the TRIM (Tripartite motif) or RBCC (RING, B-Box, coiled-coil) protein family and Vichi et ai, (2005) demonstrated E3 ubiquitin ligase activity in the N-terminal part of the molecule. 

PH Domain. The pleckstrin homology (PH) domain was required for membrane association of cytohesin-1 via phosphoinositide binding (Nagel et al., 1998). L-o-phosphatidyl-L-serine (PS) is included in the routine GEP assays described here for cytohesins as well other GEPs (Pacheco-Rodriguez et al., 1998). With or without PS, the See domain of cytohesin-1 accelerated GTP7S binding to nonmyristoylated ARF, which lacks the propensity to associate with membranes that is characteristic of native ARF. Because both ARFl and cytohesin-1 appear to require membrane... 

C Terminus. The cytohesins differ most in structure near the C-terminus. Cytohesin-1 was phosphorylated by protein kinase C at serine 394 and threonine 395, which influenced its interaction with the cytoskele-ton, but had no effect on binding to phospholipid membranes (Dierks et al., 2001). In contrast, phosphorylation of a residue at the C-terminus of cytohesin-2 resulted in lack of association with membranes and inefficient ARF activation (Santy et al., 1999). It seems likely that these, and perhaps other phosphorylations, have a role(s) in cytohesin function. 

When overexpressed in COS-7 cells, myc-cytohesin-lwas found associated with cytoskeletal structures and in the nucleus (Vitale et al., 2000). The N-terminus of cytohesin-1 appeared to be responsible for its presence in Golgi structures (Lee et al., 2000). Overexpressed cytohesin-1 was associated with the plasma membrane in Jurkat cells (Kolanus et al., 1996). This seemed to be influenced by stimuli like EGF and NGF, as well as integrin interactions with extracellular molecules such as ICAM (Kolanus et al., 1996 Venkateswarlu et al, 1999). 

To quantify GEP activity of cytohesins in vitro, most assays measure ARF binding of guanine nucleotides using either radiolabeled nucleotides (e.g., Pacheco-Rodriguez et al., 1998) or changes of the tryptophan fluorescence of ARF that occur when bound GDP is replaced by GTP (e.g., Beraud-Dufour et al., 1998). GEP action on ARFs in cells has been evaluated by pull-down assays to recover activated ARF bound to GGA proteins (Santy and Casanova, 2001). 

One of the confounding technical problems, especially with recombinant proteins, is the presence of improperly folded molecules with only a small, usually unknown and difficult to quantify, fraction of active protein. In an attempt to minimize this problem in practice, we induce at least 10 different colonies containing the cytohesin-1 construct and pool the cells for protein purification, which improves the assay reproducibility. In addition, assays with native ARFl/3 are routinely included with those employing recombinant preparations (Fig. 3) (Pacheco-Rodriguez et al., 1999, 2002). Some of the Sec7 domain proteins and especially certain recombinant fragments are unstable under assay conditions (Morinaga et al., 1999 Sata et al., 1999). For any quantitative evaluation of GEP... 

Fig. 3. Effect of cytohesin-1 or its Sec7 domain on pS]GTP7S binding by native ARFl/3. A partially purified preparation of ARFl and ARF3 was incubated for the indicated time without ( ) or with recombinant cytohesin-1 ( ) or its Sec7 domain (A) (reprinted with permission from Pacheco-Rodriguez et al, 1999).

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