Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Rab Geranylgeranyltransferase

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101635

Synonyms

Historical Background

Many proteins interact with biological membranes in a transient way, and such interaction is a prerequisite of numerous cellular processes and vesicular transport, among others. Posttranslational lipid modification of a polypeptide results in a covalent attachment of the hydrophobic anchor and increased affinity of the particular peptide to cellular membranes. Two kinds of lipid moieties are most often added to intracellular proteins: fatty acids or prenyl groups (reviewed in Wang and Casey 2016; Hentschel et al. 2016). Protein geranylgeranylation is the posttranslational modification leading to an attachment of a 20-carbon geranylgeranyl isoprenoid chain to a specific cysteine residue in a protein by a thioether bond. Rab geranylgeranyltransferase (RGGT, EC 2.5.1.60) is an enzyme responsible for di-geranylgeranylation of Rab proteins. The activity of this enzyme was first described in 1992 in rat brain cytosol (Seabra et al. 1992) and was later found in all eukaryotes (reviewed in Gutkowska and Swiezewska 2012). The RGGT complex is built of three subunits: α and β subunits forming a catalytic core and an accessory subunit Rab escort protein (REP) responsible for presentation of the protein substrate to the αβ dimer. In mammals α and β subunits of RGGT are encoded by single genes while in contrast REP is encoded, quite uniquely among organisms, by two genes CHM and CHML (Rasteiro and Pereira-Leal 2007). The enzyme recognizes cysteines localized in a conserved motif CXCX, CCXX, or XXCC at the C-terminus of the polypeptide (Fig. 1), exclusively in Rab protein context (Seabra et al. 1992). The di- geranylgeranylation enables appropriate localization of Rab proteins within the cell. Non-geranylgeranylated Rabs localize to the cytoplasm, while mono-geranylgeranylated are stored in the ER. Importantly, at both these localizations, they are unable to perform their normal functions in vesicle budding, transport, and fusion (Gomes et al. 2003). To illustrate the role of this lipid modification, phase partitioning of the peptide analogues of Rabs C-termini was analyzed in vitro using the liposomal membranes. Di-geranylgeranylated polypeptides show slow rates of spontaneous partitioning “off” lipid membrane, about 10–50 h, in contrast to mono-geranylgeranylated, that dissociate in seconds or less – similarly to a 14-carbon fatty acid modification (Silvius and l’Heureux 1994; Shahinian and Silvius 1995). Geranylgeranyl anchors promote association of lipids such as diacylglycerols and 3–4-phosphoinositides in the vicinity of Rabs (Fratti et al. 2004). Interestingly, GG-modified Rabs are excluded from cholesterol and sphingolipid-enriched membrane microdomains that have an ordered liquid phase properties (Keller and Simons 1998; Melkonian and Ostermeyer 1999). Deficiency of RGGT results in hereditary diseases, e.g., human choroideremia (retinal dystrophy) (Seabra et al. 1993) or mice gunmetal (thrombocytopenia with reduced platelet alpha and delta granule content) (Detter et al. 2000; see below). Crystal structure of rat RGGT was reported (Zhang et al. 2000; Pylypenko et al. 2003), and the mechanisms of its enzymatic activity were proposed (Thoma et al. 2001a; Baron and Seabra 2008).
Rab Geranylgeranyltransferase, Fig. 1

Scheme of Rab geranylgeranylation on two adjacent C-terminal cysteine residues

RGGT Structure

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

Two models of Rab prenylation were proposed based on the crystal structures and kinetic data. In the first model (Thoma et al. 2001a; Guo et al. 2008), REP initially recognizes Rab and presents it to a preformed catalytic RGGT heterodimer. The order of prenylation of the two cysteines is random, but the mono-geranylgeranylated product of the first reaction does not dissociate from the enzyme. The lack of precise positioning and high affinity of the enzyme-substrate interaction may be the cause why RGGT is considered as “slow” protein prenyltransferase (K1 = 0.16/s, K2 = 0.04/s). Following the second prenylation, the high affinity binding of the next molecule of the geranylgeranyl diphosphate substrate dislodges the bulky di-geranylgeranylated peptide product from the active site. Consequently, the di-geranylgeranylated C-terminus of the Rab molecule associates with the lipid binding site of REP. This induces REP conformational change and liberates the RGGT heterodimer from Rab-REP complex. Binding of the Rab-REP complex to mammalian RGGT was found to be strongly modulated by GGPP (Thoma et al. 2001b). The affinity of RGGT for the unprenylated Rab-REP complex increases from 120 to 2 nM in the presence of GGPP. Affinity of di-geranylgeranylated Rab-REP complex to RGGT was 2 and 18 nM in the absence and presence of the GGPP, respectively (Fig. 2a).
Rab Geranylgeranyltransferase, Fig. 2

Two models of Rab geranylgeranyltransferase reaction. (a) RGGT catalytic heterodimer recognizes preformed REP-unprenylated Rab complex. GGPP binding elevates the affinity of RGGT to REP-Rab. Both steps of geranylgeranyl addition to Rab proceed without Rab dissociation from the enzyme. Binding of the next GGPP molecule to RGGT dimer lowers the {REP-di-geranylgeranylated Rab}-RGGT affinity and liberates REP-prenylated Rab complex. REP escorts Rab to the membrane (Thoma et al. 2001a). (b) RGGT in the presence of GGPP forms complex with REP and only afterwards unprenylated Rab binds to the enzyme. Further steps of RGGT cycle proceed the same way as described in the first model (Baron and Seabra 2008)

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).

Summary

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.

See Also

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Institute of Biochemistry and Biophysics, Polish Academy of SciencesWarsawPoland