The rat α subunit of RGGT (RGGTA) is very similar to the corresponding α subunit of the two other protein prenyltransferases which catalyze similar reaction although do not accept Rab peptide as a substrate, i.e., protein farnesyltransferase (FT) and protein geranylgeranyltransferase (GGT I), containing 15 α-helices arranged in a crescent-shaped structure enveloping the beta-subunit (Zhang et al. 2000). Structurally it can be classified as a tetratricopeptide repeat (TPR) superfamily protein. RGGT α subunit interacts with REP on one surface and with β subunit of RGGT (RGGTB) on the opposite surface of the protein (Pylypenko et al. 2003). The RGGT α/REP interaction is allosterically regulated by binding of phosphoisoprenoid substrate, geranylgeranyl diphosphate (GGPP) (Thoma et al. 2001b; Pylypenko et al. 2003). Rat RGGT α displays a globular C2 domain inserted in the middle of the TPR domain. The function of this insertion is unclear, although it is not involved in the formation of complexes either with REP or Rabs (Pylypenko et al. 2003). At the C-terminus of the rat RGGT α subunit, a leucine-rich repeat (LRR) domain is present, not found in the related protein farnesyltransferase. RGGT α truncated of LRR and C2 domains is stable in a dimer with RGGT β subunit and shows structure and prenylation activity comparable to an intact wild-type enzyme (Guo et al. 2008).
The β subunit of RGGT (RGGTB) of rat is an α-α barrel composed of 12 α-helices, resembling the fold of the β subunits of protein farnesyltransferase (FT) and geranylgeranyltransferase I (GGT I) (Zhang et al. 2000). Only one geranylgeranyl diphosphate binding site per RGGT β is present (Guo et al. 2008).
REP belongs to the same family as Rab GDP dissociation inhibitor (RabGDI), and the two proteins are very similar in structure (Alory and Balch 2003). Large regions of REP are predicted to be disordered, and probably these parts of protein are natively unfolded (Rasteiro and Pereira-Leal 2007). They might have a regulatory function. Similarly, the very C-terminus of REP is disordered in the structure of REP and RGGT heterodimer in the absence of Rab (Pylypenko et al. 2003). The specificity of the enzymatic complex toward Rabs is confirmed by REP-Rab and REP-RGGT dimer interaction and not by the direct binding of Rab C-terminus to the catalytic heterodimer (Anant et al. 1998).
RGGT Mode of Action
According to the alternative model of an enzymatic complex formation (Baron and Seabra 2008), unprenylated Rab binds to the preformed ternary complex of REP and RGGT αβ heterodimer. In this model GGPP binds to RGGT dimer at a first step and enhances its affinity for REP. Only this full enzymatic complex bound to GGPP attracts unprenylated Rab. Similarly as above, new GGPP molecule promotes dissociation of di-geranylgeranylated Rab-REP from RGGT dimer (Fig. 2b). Application of many functionalized analogues of geranylgeranyl moiety enabled in vivo and in vitro analysis of enzymatic properties of Rab geranylgeranyltransferase as well as localization and prenylation status of Rab proteins within animal cells (reviewed in Wang and Distefano 2016).
Role of RGGT in Pathogenesis
Information on hereditary monogenic diseases caused by deficiency of Rab geranylgeranylation has recently been summarized (Mitra et al. 2011; Hutagalung and Novick 2011 and references therein). The best-studied animal model revealing the consequences of RGGT mutation is gunmetal mutation in mice RGGT α encoding gene (G→A substitution in a splice acceptor site) (Detter et al. 2000). RGGT activity and RGGT α protein levels are reduced fourfold in gunmetal platelets, and Rab27b protein is particularly hypoprenylated in platelet precursor cells. The phenotype of the homozygous gunmetal mice manifests as prolonged bleeding caused by defects in platelets and megakaryocytes, macrothrombocytopenia, and reduced platelet α- and δ-granule content (storage pool deficiency), and the animals also have partial cutaneous albinism (Detter et al. 2000). Moreover, cytotoxic T-lymphocyte ultrastructure and function are impaired in gunmetal mice due to Rab27a and other Rab species hypoprenylation (Stinchcombe et al. 2001). Impaired prenylation of Rab GTPases in the gunmetal mice causes defects in bone cell (osteoclasts and osteoblasts) function (Taylor et al. 2011). Based on phenotypic similarities gunmetal mutant is sometimes considered as a model of the human Hermansky-Pudlak syndrome (symptoms include albinism, bleeding tendency, and lung disease); however, definite arguments supporting this concept are still missing. Hypoprenylation of Rab38 protein in lung cells of chocolate mice causes abnormal lung surfactant homeostasis and aberrant alveolar structure (Osanai et al. 2008), resembling some manifestations of Hermansky-Pudlak syndrome as well. In many cases of human Griscelli syndrome type 2, mutations causing premature stop codons and loss of geranylgeranylation motif in Rab27a lead to partial albinism (silver gray hair) due to mistargeting of melanosomes in hair shaft. Severe immunological symptoms (due to abnormal granule release within cytotoxic T lymphocytes) often leading to death in infancy are also characteristic for this rare disease (van Gele et al. 2009). Similarly in human Carpenter syndrome, hypoprenylation of Rab23 caused by C-terminal protein truncations and loss of prenylatable cysteines leads to multiple defects, such as skull deformations, polydactyly, and heart defects (Wang et al. 2006).
Geranylgeranylated Rab proteins are highly abundant in cells and tissues with active vesicular transport. The role of Rab in oncogenesis is broadly discussed since aberrant endocytosis, vesicle targeting, and receptor recycling are involved in altering cell adhesion, migration, proliferation, polarity, asymmetrical division, and overall survival (reviewed in Recchi and Seabra 2012). Cancer cells are particularly dependent on active endocytosis of nutrients, hormones, and growth factors. They also actively reorganize their shape in the process of metastasis and secrete substances needed for matrix digestion or vessel formation. Indeed overexpression of Rab proteins has been found in many cancers, in many cases correlating with poor prognosis for the patient. Recently, specific inhibitors of RGGT, such as bisphosphonates (e.g., 3-PEHPC), have been used to treat myeloma cultured cells (Roelofs et al. 2006), and combined therapy by dual specificity GGT I and RGGT inhibitor GGTI-2Z and statins resulted in inhibition of proliferation and cell cycle arrest of peripheral nerve sheath tumor and human breast ductal carcinoma cell lines (Sane et al. 2010). Peptidomimetic strategy of RGGT inhibitor design seems also to be promising (Tan et al. 2009) as well as using derivatives of a natural compound, psoromic acid (Deraeve et al. 2012).
Inhibitors of RGGT may be found useful for treatment of bone diseases characterized by extensive resorption, such as osteoporosis (Itzstein et al. 2011); moreover, siRNA treatment against RGGT or REP in pancreatic islet β cell line markedly attenuated glucose-stimulated insulin secretion (Arora et al. 2012). In addition 3-PEHPC shows activity against systemic lupus erythematosus in mice model (Caza et al. 2014).
In summary, RGGT seems a promising target in therapy of cancer and serious chronic diseases, although the gaps in basic knowledge on regulation of the enzyme activity and specificity remain to be filled. Numerous questions appeal for immediate answer. Do additional domains inserted in the TPR structure of RGGT α subunit have a regulatory role? Which proteins or lipids do they interact with? Does mammalian REP, similarly to its yeast counterpart, play a role in nutrient sensing and switching on and off the TOR kinase pathway? What is the hierarchy of Rab prenylation in particular cells and tissues? Can it be engineered in order to help alleviate serious hereditary diseases’ symptoms? Discovery of new RGGT inhibitors and geranylgeranyl analogues as well as introduction of gene therapy (as in the case of choroideremia disease) will enable to answer these questions in the coming years.
- Wang YC, Distefano MD. Synthetic isoprenoid analogues for the study of prenylated proteins: Fluorescent imaging and proteomic applications. Bioorg Chem. 2016 Feb;64:59-65. doi: 10.1016/j.bioorg.2015.12.003.Google Scholar