The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.
- membrane trafficking
- pathogen infection
The Rab proteins belong to the Ras small GTPase superfamily. During the past two decades, a large amount of literature has addressed the properties and functions of the Rab proteins and established Rab GTPases as master regulators in membrane trafficking [1–5]. Rab exerts its function through the GTPase cycle. Newly synthesized Rab is recognized by REP (Rab escort protein) and transferred to RabGGT (Rab geranylgeranyl transferase) for prenylation, the prenylated Rab then goes on to the GTPase cycle. In the cytosoplasm, GDP-bound Rab is associated with GDI (GDP dissociation inhibitor), and GDF (GDI displacement factor) recruits Rab to the membrane where GEF (guanine-nucleotide-exchange factor) converts it into the GTP-bound active form, which interacts with downstream effectors to exert its biological functions. GTP hydrolysis of Rab converts it back into the GDP-bound form, which is generally facilitated by GAP (GTPase-activation protein). It has been indicated that Rab proteins regulate not only membrane trafficking, but also cell signalling, cell growth, cell survival and development [6,7]. Rab proteins and their associated regulators or effectors have been implicated in many diseases, such as cancer, pigmentation disorder, neuropathy and lipid metabolism disorders. Genetic mutations and abnormal expression of Rab proteins or their partners are closely linked to disease pathogenesis [8–13]. Furthermore, pathogens usually hijack Rab-mediated trafficking machineries in host cells for infection and survival [10,14]. Therefore it will be very useful to develop therapeutic strategies targeting Rab or modulating Rab-mediated membrane traffic .
For the Rab family of GTPases, approx. 70 members have been identified in mammals. Rab7 is one of Rab proteins which has been investigated extensively. The extensive studies have revealed that Rab7 is a central factor in endosomal membrane trafficking. Trafficking disorders, resulting from mutation or dysfunction of Rab7, can cause human diseases, and the Rab7-mediated membrane traffic process is linked to pathogen infection and survival. In the following sections, we give a brief review on the functions of Rab7, summarize the involvement of Rab7 in disease pathogenesis and discuss the possible underlying mechanisms that are regulated by Rab7.
RAB7: CENTRAL ROLES IN THE ENDOCYTIC PATHWAY
Rab7 is associated primarily with late endosomal structures, and perhaps it is the only lysosomal Rab protein found to date[16–20]. The functions of Rab7 have been investigated extensively. Feng et al.  indicated that the dominant-negative mutants Rab7-T22N and Rab7-N125I blocked trafficking of the VSV (vesicular stomatitis virus) G protein from the early endosome to the late endosome, without affecting its internalization from the surface. In addition, the Rab7 mutants caused accumulation of cathepsin D and the cation-independent mannose-6-phosphate receptor in early endocytic compartments, and inhibited the maturation process of cathepsin D . Vitelli et al.  examined the effects of Rab7 on degradation of internalized LDLs (low-density lipoproteins) and found that dominant-negative forms of Rab7 inhibited LDL degradation. These studies conclude that Rab7 serves as a key factor in regulating transport of lysosome-destined enzymes and internalized surface proteins to the lysosome through the endocytic pathway.
It has been observed that Rab proteins act in concert with their special tethering complexes to determine a unique membrane identity, which generates various Rab-defined membrane domains [2,4,24,25]. Within the endocytic pathway, many Rab proteins localize to endocytic compartments: Rab4, Rab5, Rab11, Rab22 and Rab25 are primarily associated with the early and recycling endosomes [26–30]; Rab9 and Rab7 are localized to late endosome [31,32]. Rab7 is additionally localized to the lysosome and was thus characterized also as a lysosome-associated Rab protein . Rab5 and its effectors, rabaptin-5, Rabex-5, EEA1 (early endosome antigen 1), Rabenosyn-5, hVps34 (human vacuolar protein sorting 34)/p150 may be defined as an early endosomal membrane domain . Rab4 and Rab11 have been shown to associate with the exocyst complex at the recycling endosomal membrane . Rab7 and its effectors are emerging to generate Rab7-defined membrane domains, playing central roles in the late endocytic pathway (Figure 1). Rab7-defined membrane domains include late endosome, intermediate hybrids of late endosomes–lysosomes, and the lysosome. Rab7 regulates late endosomal membrane fusion and trafficking mediated by a tethering complex, which is carried out by the HOPS (homotypic fusion and protein sorting; also refers to class C Vps protein) complex. The HOPS complex consists of Vps11, Vps16, Vps18, Vps33, Vps39 and Vps41. Vps39 binds to Rab7 and exhibits GEF activity for Rab7. Vps33 is a munc-1-like protein, which can associate with the endosomal SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein to regulate membrane fusion, thus Rab7 interacts with the HOPS complex and is recruited to the endosomal membrane to regulate vesicle fusion [this process may also require phosphoinositides regulated by PI3K (phosphoinositide 3-kinase)/hVps34] [20,35–39]. The distribution and movement of the late endosome/lysosome is regulated by interaction of Rab7—RILP–dynein–dynactin (where RILP is Rab7-interacting lysosomal protein) [40,41].
Rab5-defined early endosomal membrane domains and Rab7-defined late endosomal/lysosomal membrane domains do not work separately, but sequentially and dynamically to co-operate in the endocytic pathway. Along the endocytic pathway, cargos are internalized from the plasma membrane and transported to the early endosome. At the early endosome, cargos are sorted to different destinations: the recycling endosome, the late endosome and lysosome or the Golgi apparatus (Figure 1), in which the Rab cascade determines the unique trafficking pathway [24,42]. Membrane trafficking from the early endosome to the late endosome is determined by the recruitment of Rab5 to early endosomes and, sequentially, acquisition of Rab7 followed by loss of Rab5 in the late endosomes. By employing live-cell imaging technology, Rink et al.  observed the conversion of Rab from Rab5 into Rab7 in endosomal membrane dynamics during the transport from the early endosome to the late endosome. The dissociation of Rab5 and subsequent recruitment of Rab7 were regulated by the HOPS complex. The HOPS complex may play a critical role in this Rab cascade, since it is also an effector of Rab5 GTPase . The sequential action of Rab5 and Rab7 in endocytosis and endosomal sorting/maturation was also observed in Xenopus oocytes  and in axonal retrograde transport in neurons . Rab7, working co-operatively with Rab5, was investigated in the recruitment of the retromer complex [comprising SNX1/2 (sorting nexin 1/2), Vps26, Vps29 and Vps35]. The interaction between Rab7 and the retromer complex has been studied in the protozoan parasite Entamoeba histolytica . Recently, the works by Rojas et al.  offered a possible mechanism for Rab5 and Rab7 to regulate retromer recruitment and function. In this model, Rab5 interacted with PI3K and recruited SNX1/2 to membrane, whereas Rab7 recruited the retromer core complex Vps26–Vps29–Vps35 through direct interaction with Vps26. The following interaction of SNX1/2 with Vps26/29/35 mediates cargo transport. Unexpectedly, the role of RILP in this process was not examined, and the HOPS complex may also play a role in this regulation. The involvement of Rab7 in retromer regulation suggests that Rab7 also participates in retrograde transport between the endosomes and the Golgi apparatus.
The functions of Rab7 are regulated via the GTPase cycle and its partners, including upstream regulators and specific downstream effectors, as shown in Figure 2. So far, there are no upstream regulators, such as REP, RabGGT, GDI or GDF, identified specifically for Rab7, and Rab7 may share these common regulators with other Rab proteins. For example, Rab7 prenylation is regulated by REP-1 . Similar to other GTPases, Rab7 may have its specific GAP and GEF. The yeast protein Gyp7p and its mammalian homologue TBC1D15 [a TBC (Tre2/Bub2/Cdc16)-domain-containing protein] were identified as GAPs for Ypt7 and Rab7 respectively [49,50]. The HOPS complex exhibits GEF activity for Rab7 [37,38].
As shown in Table 1, Rab7 interacts with multiple downstream effectors. RILP is one of the well-studied Rab7 downstream effectors; the Rab7–RILP interaction is a crucial mechanism in regulating endosomal traffic and biogenesis of late endosomal/lysosomal compartments [40,51]. Overexpression of RILP caused enlarged Rab7-containing late endosomes/lysosomes with a peri-nuclear distribution. The truncated form of RILP also impaired endosomal transport of EGFR (epidermal growth factor receptor) and LDL receptors . Further investigation revealed that Rab7 regulates lysosomal movement towards the MTOC (microtubule-organizing centre) through RILP to interact with the minus-end-directed motor protein dynein–dynactin complex, with the participation of another Rab7-interacting effector ORP1L [OSBP (oxysterol-binding protein)-related protein 1] [41,52]. RILP can also interact with Vps22 and Vps36 to regulate the late endosomal or MVBs (multivesicular bodies) degradation pathway [53,54]. Other effectors for Rab7 have also been described (Table 1). Rabring7 (Rab7-interacting RING-finger protein) is involved in EGF degradation as an E3 ligase . PI3K/hVps34/p150 forms complexes with Rab7, suggesting that Rab7 may also regulate PI3K activity and membrane trafficking from the early endosome to the late endosome . Rab7 can bind to the proteasome α-subunit XAPC7 and recruit it to the late endosome. XAPC7 may serve as a negative regulator for Rab7, since overexpression of XAPC7 impairs EGFR-mediated endocytosis, which can be rescued by expressing wild-type Rab7 . Rab7 directly interacts with small GTPase Rac1 to regulate the formation of RBs (ruffled borders) in osteoclasts . Collectively, Rab7 interacts with multiple effectors and participates in multiple biological events.
Rab7 plays a central role, not only in endosomal traffic, but also in many other cellular and physiological events, such as growth-factor-mediated cell signalling, nutrient-transportor-mediated nutrient uptake, neurotrophin transport in the axons of neurons and lipid metabolism. In addition to the normal endocytic traffic pathway, Rab7 is involved in regulation of some specialized endosomal membrane trafficking, such as maturation of melanosomes, pathogen-induced phagosomes (or vacuoles) and autophagosomes. In the following sections, we will describe some diseases or physiological disorders caused by mutations or dysfunction of Rab7, and how Rab7 and its partners are engaged in infectious diseases caused by microbial pathogens or viruses.
DISEASES CAUSED BY MUTATIONS OR DYSFUNCTION OF RAB7
Rab proteins are master regulators of membrane trafficking. Studies have linked Rab proteins and Rab-regulated traffic to many diseases [10,13]. For examples, genetic defects in Rab27a and its partner Myo5a cause Griscelli syndrome ; mutations in the upstream regulators of Rab, REP, RabGDI and RabGGT, are linked to the renal degeneration disease choroidaermia, X-linked mental retardation and HPS (Hermanskey–Pudlak syndrome) respectively [60–62]; abnormal expression of Rab25 is associated with the development of ovary and breast cancer . A number of studies have indicated that Rab7 is another important Rab involved in disease pathogenesis. Mutations in Rab7 gene or dysfunction of Rab7 and Rab7-interacting effectors may cause diseases or physiological disorders (Table 2).
Rab7 in neuropathy
Several Rab proteins are expressed in neurons and glia, and some of them are closely related to neurological functions . There are reports showing that defects of Rab5 and Rab7-regulated late endocytic traffic are related to neurological diseases, such as Alzheimer's disease and Down's syndrome . The direct evidence that Rab7 is involved in neuropathy comes from the studies on CMT2B (Charcot–Marie–Tooth syndrome type 2B). Mutations in Rab7 are well characterized as genetic defect markers for CMT2B, which is part of a group knowns as the HSNs [hereditary sensory neuropathies; also referred as HSANs (hereditary sensory and autonomic neuropathies)]. Patients suffering this defect exhibit progressively neurological disorders with clinical symptoms of distal sensory loss, muscle weakness and foot ulcerations [66–69]. So far, four mutations in Rab7 have been identified in four different families with CMT2B, and all mutations occur in the conserved amino-acid residues adjacent to the switch II region in the C-terminal part of Rab7, including the mutations L129F, K157N, N161T and V162M [70–73].
Recently, the underlying mechanisms for CMT2B due to Rab7 mutations have been investigated. Spinosa et al.  studied the biochemical and functional properties of three Rab7 mutants, Rab7-L129F, Rab7-N161T and Rab7-V162M, and found all three mutant forms exhibited lower GTPase activity than the wild-type form and had a preference for GTP binding, indicating the mutant forms of Rab7 are more activated than wild-type Rab7, which is similar to the constitutively active mutant Rab7-Q67L, and all the mutants can interact with the downstream effector RILP . Similar properties were also studied on the Rab7-K157N mutant . The results suggest that activated Rab7 and Rab7-regulated endocytic traffic processes may be responsible for CMT2B neuropathies. Interestingly, another type of HSAN, HSAN-1 is due to the mutation in the gene for SPTLC1 (serine palmitoyltransferase long chain), which is involved in sphingolipid synthesis [69,75]. Since Rab7 is also involved in the transport of sphingolipids, CMT2B and HSAN-1 may potentially share an overlapping pathogenesis mechanism.
Rab7 regulates membrane trafficking in neuronal cells. In PC12 cells, Rab7 can associate with the NGF (nerve growth factor) receptor TrkA (tropomyosin receptor tyrosine kinase A) at endosomes. Inhibiting Rab7 activity resulted in accumulation of TrkA in the endosome and potentiated NGF-stimulated signalling of TrkA to induce neurite outgrowth . Axonal transport contributes long-range communication in neurons and is essential for the survival and differentiation of neurons. Using TeNT Hc (atoxic fragments of the tetanus neurotoxin) as a marker, which shares the same retrograde pathway as neurotrophins and their receptors, Deinhardt et al.  demonstrated that functional Rab7 is required for retrograde transport of the neurotrophin receptor . The above data suggest that dysfunction of Rab7 and the disruption of Rab7-regulated membrane traffic may inhibit neuron growth or promote apoptosis due to nutrient deficit, causing neurodegenerative diseases. Rab7 controls endosomal trafficking of TrkA and TrkA-mediated neuritogenic signalling, and also regulates axonal retrograde transport of neurotrophin. These results may also partly explain how Rab7-regulated membrane traffic is responsible for CMT2B and other neurodegenerative diseases. The reason why mutations of ubiquitously expressed Rab7 have a more profound effect on peripheral neurons, with little effects on other tissue/organs, may be due to the requirement of more-tightly regulated membrane trafficking in neurons.
Rab7 in cancer and cell survival
Aberrant endocytosis and altered lysosomal function result in defective growth-factor transport and unbalanced levels of surface proteins, such as integrins and E-cadherin, leading to tumorigenesis and cancer metastasis [77,78]. Rab GTPases, as master regulators in membrane traffic, are proved to be involved in cancer development . Rab25 is a well-established tumorigenesis-associated Rab and is highly homologous to Rab11, and endogenously overexpressed in most ovarian and breast cancer samples in a constitutively active form, which is unique among Rab proteins. Cheng et al.  provided data indicating that overexpression of Rab25 promotes cell transformation, inhibits apoptosis and induces tumour progression, probably through the PI3K/AKT signalling pathway. Rab25 may also be related to other cancer such as OC/PPC (ovarian/primary peritoneal serous carcinoma) and prostate cancer .
The results from Croizet-Berger et al.  showed that thyroid hormone production was regulated by Rab5a and Rab7. cAMP stimulation elevated the expression of Rab5a and Rab7 in adenomas, linking Rab7 to the formation of benign thyroid autonomous adenomas . Davidson et al.  also found Rab7 is overexpressed in DMPM (diffuse peritoneal malignant mesothelioma). In addition, v-Src induces activation of Rab7, which may be related to epithelial-to-mesenchymal transition during tumour progression . Studies by Edinger et al. [81,82] indicate that Rab7 is involved in a cell survival pathway. Upon growth-factor depletion, Rab7 down-regulates surface nutrient transporters through endocytic degradation, preventing growth-factor-independent survival, but inhibition of Rab7 sustains surface nutrient transporters, thus promoting long-term cell survival, which is dependent on the AKT survival signalling pathway. Furthermore, Edinger and colleagues [81,82] demonstrated that inhibition of Rab7 co-operated with the adenoviral E1A protein to promote transformation of p53−/− MEFs (mouse embryonic fibroblasts), thus Rab7 was proposed to act as a potential tumour suppressor (reviewed in ). However, there is insufficient evidence to conclude that Rab7 functions as a tumour suppressor. As mentioned above, Rab7 is actually overexpressed in some cancer cells or tissues, as described previously [79,80], and the transformation effects of dominant-negative Rab7 required the crucial help of the E1A protein and the absence of p53 in the studies by Edinger and colleagues [81,82], and these studies were carried out under nutrient starvation condition which may differ slightly from the environmental conditions for tumorigenesis that are usually rich in growth factors. Lackner et al.  provided another view on the function of Rab7 in apoptosis. Inhibiting the upstream regulator RabGGT prominently induces apoptosis of germ cells in Caenorhabditis elegans and mammalian cancer cells. Lackner et al.  also examined the effects of knockdown of Rab5, Rab7 and components of the HOPS complex by RNA interference in C. elegans, and found that knockdown of both Rab proteins promoted germ cells apoptosis. In addition, knockdown of the HOPS complex (comprising Vps11, Vps16, Vps18, Vps33 and Vps39) also induced apoptosis, suggesting that Rab7 and the Rab7-regulated pathway are involved in suppressing apoptosis . In disagreement to the conclusion by Lackner et al. , Kinchen et al.  got similar results from knockdown of Rab5, Rab7 and the HOPS complex in C. elegans, but they examined the increase of apoptotic cells for loss of ‘cleaning’ functions by defective phagocytosis. Taken together, the underlying mechanism for cancer, cell survival and apoptosis regulated by Rab7 is still not yet understood.
Rab7 is also emerging as a regulator for the autophagic pathway, another mechanism for cell death and survival, which is related to many diseases, such as cancer and heart failure [85,86]. The autophagic process is initiated by engulfment of cytoplasmic materials into a unique membrane (phagophore) to form an autophagosome; the autophagosome then undergoes maturation through fusion with endosomal vesicles and lysosomes to form a lysoautophagosome, in which materials are degraded to provide nutrients and energy for cell survival under nutrient depletion. The late autophagic process is similar to late endocytic fusion, and the mechanism for regulating autophagosome maturation is becoming clear, and Rab7 has been shown to be a major factor in governing the transport and fusion events during maturation of autophagosome [87,88]. Furthermore, Rab7 is regulated by Beclin1, a tumour suppressor able to induce autophagy, through the Beclin1–UVRAG–HOPS complex–Rab7 interaction cascade (where UVRAG is UV-irradiation resistance-associated gene product), since the UVRAG and the HOPS complexes are effectors for Beclin1 and Rab7 respectively .
Rab7 in lipid trafficking disorders
Sphingolipids associate with cholesterol in the plasma membrane to form unique lipid rafts, which play important roles in membrane organization, cell signalling etc. . Upon stimulation, sphingolipids and cholesterol can be internalized through either clathrin-dependent or caveolin-mediated endocytosis into late endosomes. In the late endosome, the sphingolipids can be further transported to the lysosome for degradation, or the Golgi apparatus or other organelle; mis-regulation of these transport events results in accumulation of sphingolipids in late endosomes and causes SLSDs (sphingolipid storage diseases) . NPC (Niemann–Pick type C) disease is a well known SLSD, which is caused by mutations in the NPC-1 and NPC-2 genes, characterized by accumulation of sphingolipids and cholesterol in the late endosome due to a lipid traffic jam. There is evidence suggesting that Rab7 is linked to NPC disease . Zhang et al.  provided data demonstrating that NPC-1 protein is associated with the Rab7-containing late ensosome. Choudhury et al.  investigated lipid trafficking in NPC cells. Their results showed that the fluorescent glycosphingolipid, BODIPY®–lactosylceramide, is targeted to the Golgi in normal human skin fibroblast cells, and dominant-negative mutants of Rab7 and Rab9 impaired the Golgi targeting. Furthermore, overexpression of wild-type Rab7 or Rab9 (but not Rab11) can reduce cholesterol accumulation in NPC cells and restore the trafficking of BODIPY®–lactosylceramide to the Golgi, which indicates a novel potential therapeutic strategy for this disease . However, Lebrand et al.  found that overexpressing Rab7 increased the accumulation of cholesterol and reduced late endosomal mobility, which is not consistent with the study by Choudhury et al. . This difference may be due to the different cell types that were used, but more work is required to reveal the underlying mechanisms for the involvement of the regulation of Rab7 in NPC disease.
Intriguingly, accumulation of cholesterol affects APP (amyloid precursor protein) processing by inhibiting β-secretase, but enhancing γ-secretase, to produce both Aβ40 (amyloid β-peptide 40) and Aβ42, and alters presenilin localization to Rab7-positive late endosomes . As discussed above, cholesterol accumulation is also regulated by Rab7 in NPC disease cells, suggesting that Rab7 links lipid trafficking disorders to neurodegenerative disease.
Rab7 is also implicated in the tub-1 pathway to regulate fat storage. tub-1 is a transcription factor, and mutation in this gene results in adult-onset obesity, insulin resistance and progressive neurosensory deficits . Mukhopadhyay et al.  found that tub-1 interacts with the RBG-3 (RabGAP initially supposed to target Rab3) protein, which serves as a GAP for Rab7. RNA interference of Rab7 can reduce fat storage in C. elegans, proposing another role of Rab7 in lipid metabolism through interaction with RBG-3.
Although genetic defects in Rab7 have not been identified in lipid metabolism disorder, the Rab7 gene is up-regulated by a cholesterol-rich diet in the liver and atherosclerotic plaques of arteries , supportive for a role of Rab7 in diseases related to lipid trafficking disorders. However, the underlying mechanisms for the regulation of Rab7 in lipid trafficking remain to be elucidated. In particular, little is known about the interacting partners for Rab7 that are involved in these regulations. Investigations of the interaction between Rab7 and ORP1L (a member of the human OSBP family involved in cholesterol and sphingomyelin metabolism) may provide one of the starting clues for studying these mechanisms .
Rab7 in osteoclast function
Bone-resorbing osteoclasts are highly polarized with distinct membrane domains: SZ (sealing zone), RB, BD (basolateral domain) and FSD (functional secretory domain). The resorption process includes: resorption of broken bone matrix in RBs, transcytosis of degraded materials and secretion in FSDs. Disruption of bone resorption results in osteopetrosis, but excessive resorption induces osteoporosis. The RB is a unique structure that is similar to late endosomes/lysosomes and is characterized by acidic environments, association with Lamp1 (lysosome-associated membrane protein 1) and Lamp2 etc. The RB is crucial for proper bone resorption. The function of the RB depends on vesicular trafficking regulated by Rab GTPases , and Rab7 is one of most important Rab GTPases in regulating osteoclast function. Rab7 is found highly expressed in bone-resorbing osteoclasts and predominantly localized to the RB. Decreasing expression level of Rab7 disrupted the polarization of osteoclasts and impaired bone resorption in vitro . The more profound regulation mechanisms were investigated more recently. Sun et al.  identified Rac1 as a Rab7-interacting effector, and the Rab7–Rac1 interaction as being regulated by the formation of RBs in osteoclasts. Furthermore, the Rab7–Rac1 interaction suggests that Rab7 regulates membrane trafficking, which is orchestrated by the interactions between RLIP–dynein–dynactin–microtubules and Rac1–actin filaments. In addition, a pleckstrin-homology-domain-containing protein, Plekhm1, was also characterized as a potential effector for Rab7; Plekhm1 association with Rab7 is dependent of the prenylation of Rab7. Loss of function of Plekhm1 is responsible for osteopetrosis . As Rab7 plays important roles in osteoclasts, the modulation of Rab7 activity may be developed into new therapeutic strategy for treating osteopetrosis or osteoporosis.
Rab7 in the pathogenesis of other diseases
The melanosome is an LRO (lysosome-related organelle), and tyrosinase and TRPs (tyrosinase-related proteins) are melanomal membrane-bound proteins that are only expressed in melanocytes . Hirosaki et al.  found that a dominant-negative mutant of Rab7 impaired the vesicular transport of tyrosinase and TRPs from the Golgi to the melanosome, suggesting that Rab7 is involved in the biogenesis of melanosomes. However, genetic mutations in Rab7 were not observed in diseases caused by abnormal melanogenesis, such as OCAs (oculocutaneous albinisms) . However, the HOPS complex, which serves as an effector and a GEF for Rab7, plays significant roles in melanogenesis, and dysfunction of the HOPS complex results in aberrant pigmentation, albinisms and immuodeficiency disease, such as HPS; for example, reduced expression of Vps11 causes less pigmentation in medaka fish , defects in Vps18 and Vps39 induce hypopigmentation in zebrafish [108,109], Vps16 is required for endosomal trafficking and pigment-granule biogenesis in Drosophila , and the mouse model exhibiting the HPS phenotype results from a mutation in Vps33a . Furthermore, another component of the HOPS complex, Vps41, regulates alkaline phosphatase transport through interaction with the δ-subunit of the AP-3 adaptor, which is well characterized as being associated with melanogenic diseases . Gissen et al.  reported that mutation in Vps33B (Vps33a isoform) causes a severe autosomal recessive multisystem disorder, which is known as ARC (arthrogryposis-renal dysfunction-cholestasis) syndrome. In conclusion, Rab7 is likely to be involved in melanogenic diseases through interaction with its partners, such as the HOPS complex.
RAB7 IN INFECTIOUS DISEASES
In addition to diseases resulting directly from dysfunction of Rab7 or its partners, Rab7 and its partners are important factors in the pathogenesis of infectious diseases caused by micro-organisms, in which Rab7 is a key regulator in the process of phagosome maturation [114–118]. When microbial pathogens are engulfed by host cells (e.g. macrophages), they reside in a membrane-bound vacuole or phagosome; the vacuole/phagosome then fuses with late endosome to form a phagolysosome, and within the phagolysosome the pathogens are degraded. However, many microbial pathogens have evolved elaborate mechanisms to evade degradation and therefore survive within the host cells. The modulation of the function of Rab GTPases is one of the important strategies for the infection and survival of microbial pathogens . Rab7 is involved in pathogenesis of infectious diseases and has been examined in divergent microbial pathogens (Table 3), including bacteria, protozoan, fungi and viruses.
Bacteria and other microbial pathogens
Bacteria infect host cells through phagocytosis; Rab7, together with its partners (e.g. RILP), are essential factors in regulating the maturation of the phagosome into a lysophagosome, which has been well studied [119,120]. The roles of Rab7 or its partners in bacterial infection have been studied extensively for Mycobacterium tuberculosis, Mycobacterium bovis BCG, Salmonella enterica Typhimurium, Helicobacter pylori and others (Table 3).
M. tuberculosis is the most virulent pathogen in human history, causing over 1 billion people to suffer from tuberculosis. Via et al.  first established the phagosome-arrest model for pathogen survival in host cells during M. bovis BCG infection, In this model, two Rab proteins, Rab5 and Rab7, play key roles in controlling Mycobacterium phagosome maturation. The formation of Mycobacterium-containing phagosomes requires Rab5; but selective exclusion of Rab7 blocks phagosome fusion with late endosomes, and results in Mycobacterium-containing phagosome arrest in early stage, and therefore Mycobacterium can escape degradation and survive in host cells. The selective accumulation of Rab5 and exclusion of Rab7 defines the checkpoint in the mycobacterial phagosome maturation process. Interestingly, Clemens et al.  found that M. tuberculosis and Legionella pneumophila phagosomes still exhibited arrested maturation, despite acquisition of Rab7, and phagosomes containing live M. tuberculosis recruit even more active Rab7 in HeLa cells, and the authors  proposed that this discrepancy was likely due to using different cell types (macrophages and epithelial cells), different bacteria species (M. bovis BCG and M. tuberculosis) and different detection technologies. Nevertheless, subsequent investigations may provide a more mechanistic explanation for phagosome maturation arrest, even with the acquisition of Rab7 to Mycobacterium-containing phagosomes. The Rab7 downstream effector RILP is also required for phagosome maturation; the results from Sun et al.  indicated that M. bovis BCG inhibited RILP recruitment, despite Rab7 acquisition by the phagosome, therefore inhibiting phagosome maturation, in addition, Rab7 (GDP-bound form) predominates in cells infected with live M. bovis BCG, and the M. bovis BCG culture supernatant contains a factor that catalyses the GTP/GDP switch on recombinant Rab7 molecules. Previous studies  indicated that the modulation the conversion of Rab5 into Rab7 is a crucial mechanism for Mycobacterium-containing phagosome maturation arrest. A study by Roberts et al.  revealed that Rab22a, an early endosomal Rab protein, is also critical in regulating Rab7 conversion on phagosomes during M. tuberculosis infection, and Rab22a knockdown in macrophages via siRNA (small interfering RNA) enhanced the maturation of phagosomes with live Mycobacteria by increasing the association of Rab7 with phagosomes. M. tuberculosis may actively recruit and maintain Rab22a on its phagosome, thus inhibiting Rab7 acquisition and blocking phagolysosomal biogenesis . Philips et al.  reported that ESCRT (endosomal sorting complex required for transport) factors (VPS28, TGS101 and VPS4 were examined in ), as well as Rab7, restrict Mycobacterium smegmatis growth in Drosophila and mammalian cells. Because RILP interacts with the ESCRT II complex [53,54], Rab7 may regulate Mycobacteria-containing phagosome maturation through regulating ESCRT machineries in Mycobacteria infection. Taken together, Rab7 is involved in Mycobacteria infection, which is regulated by other factors.
S. enterica Typhimurium is a facultative pathogen, which invades various cell types, including epithelial cells and macrophages. Infection experiments in vitro revealed that Salmonella resides within SCVs (Salmonella-containing vacuoles) after entering into host cell. In SVCs, bacteria induce expression of SPI-2 (Salmonella pathogenicity island 2)-encoded TTSS (type III secretion system) effector SifA. SifA regulates SCV maturation into Sifs (Salmonella-induced filaments), allowing for maximal space for bacteria replication; and Sifs will not fuse with lysosomes, permitting bacteria survival and replication. The roles of Rab7 in SCVs have been investigated. Merésse et al.  showed that Rab7 is associated with SCV, and Rab7 may control the biogenesis of SCVs by recruiting lgps (lysosomal glycoproteins) to SCVs, suggesting that SCV maturation requires fusion with late endosomal membranes regulated by Rab7. Studies by Brumell et al.  indicated that Rab7 was also present in Sifs, and expression of the dominant-negative mutant Rab7-N125I inhibited Sif formation. In addition, overexpression of Rab7-N125I caused a loss of SCV integrity and increased Salmonella replication in the cytosol . Drecktrah et al.  confirmed that the acquisition of endosome/lysosome content by SCVs is Rab7 dependent using a high-resolution live-cell-imaging approach. Another study by Harrison et al.  revealed that the maturation of SCVs and Sifs is regulated by Rab7 and its effector RILP. The initial centripetal displacement of the SCV is due to recruitment of RILP by Rab7, which may govern the centripetal movement of the SCVs through interaction with dynein–dynactin complex. When Sifs are induced, RILP is depleted, despite the presence of Rab7. As a result, Sifs extend towards the periphery. In the process of Sif formation, the bacterial factor SifA is critical for disengaging of RILP from Rab7, which may serve as an interaction partner for Rab7. In summary, although many Rab proteins may associate with SCVs or Salmonella phagosome , Rab7 is probably the most important Rab protein in SCV biogenesis, in that SCV maturation requires Rab7–RILP to regulate SCV fusion with late endosomal membrane to gain some late endosomal components, such as Lamp proteins, cathepsin D and LBPA (lysobisphosphatic acid), determining SCV and Sifs as special structures different from Mycobacterium–phagosome. Nevertheless, a previous study by Hashim et al.  demonstrated that live LSPs (Salmonella-containing phagosomes) retain a significant amount of Rab5, but selectively deplete Rab7 and Rab9, with a property similar to Mycobacterium–phagosome, suggesting that different conditions in vitro, such as different cell types, bacteria strains etc., may give rise to different outcomes.
H. pylori is another representative micro-organism that possesses a survival strategy through modulating Rab7 function to escape degradation in host cells. In host cells, H. pylori releases the toxin VacA. VacA induces vacuolation, generating enlarged and peri-nuclear-distributed Helicobacter-containing vacuoles. This vacuole, containing the late endosomal markers Rab7, Lamp1 and CD63, but not Rab5, mannose-6-phosphate receptor, transferrin receptor and cathepsin D , has been described as a post-endosomal hybrid compartment, with both late endosomal and lysosomal features . Since the VacA-induced vacuoles lack the ability of degradation, bacteria can survive in this specialized structure. Rab7 was shown to be essential for the biogenesis of the VacA-induced vacuoles . The active Rab7-Q67L mutant enhances VacA-induced vacuolation, whereas Rab7-T22N or Rab7-N125I effectively inhibits vacuolation. Rab5 and Rab9 have less effect. In addition to the engagement of Rab7 in VacA-induced vacuolation, the Rab7 effector RILP is also associated with the VacA-induced vacuoles. RILP is thought to regulate large vacuole formation and cellular distribution of the VacA-induced vacuoles. Furthermore, the interaction between Rab7 and RILP is important for vacuolation, as the expression of mutant forms of RILP or Rab7 that failed to bind each other impaired the formation of this unique bacteria-containing vacuole . These data suggest that VacA prevents the maturation of the Helicobacter-containing vacuole into a bactericidal structure by retention of Rab7 and RILP. How VacA-induced vacuole maintains its unique characteristics for bacteria survival and whether VacA interacts with Rab7 or RILP (or other Rab7 effectors) remain to be answered.
The roles of Rab7 were also examined in phagocytosis of other microbial pathogens (Table 3). Brucella abortus invades host cells and resides within BCVs (Brucella-containing vacuoles). BCVs fuse with the ER (endoplasmic reticulum)-derived membrane structure to generate a replicative organelle. It has been observed that both Rab7 and RILP were recruited to the BCVs during BCV maturation. Overexpression of the dominant-negative Rab7-T22N or RILP impaired biogenesis of the ER-derived organelle and replication of bacteria , suggesting that BCV maturation requires interactions with functional late endosomal/lysosomal compartments. In phagocytosis of Bacillus anthracis spores, expression of the dominant-negative Rab7-T22N, which blocked lysosomal fusion, enhanced sterne spore survival . Exclusion of Rab7 and RILP on bacterial phagosomes in a Lamp1/Lamp2 double-knockout cell infected by Neisseria gonorrhoeae indicated that Rab7 is involved in the maturation arrest of N. gonorrhoeae-containing phagosomes . Rab7 and Rab7-Q67L localized to Coxiella burnetii-induced autophagosome-like vacuoles, suggesting that Rab7 participates in the biogenesis of this pathogen-containing vacuole . Rab7 may regulate Parachlamydia acanthamoebae trafficking along the endocytic pathway , and is probably engaged in the survival strategies by Staphylococcus aureus . Rab7 may also participate in regulating phagocytosis and the intracellular fate of conidia of the fungal pathogen Aspergillus fumigatus . Interestingly, some bacteria, such as the Crohn's disease-associated adherent–invasive Escherichia coli strain LF82, do not escape from the endocytic pathway, but undergo a normal interaction with the host endomembrane organelles, acquiring Rab7 function, and replicate within acidic and cathepsin D-positive vacuolar phagolysosomes . The mechanisms for the bacteria escaping degradation are not clear.
In protozoa infection, wild-type Leishmania donovani promastigotes inhibit phagosome maturation due to impaired recruitment of Rab7, prolonging bacterial survival in the murine macrophage cell line J774 . Rab7 was found only in the PVs (parasitophorous vacuoles) of mature BMDCs (bone-marrow-derived cells), and it was absent in immature BMDCs, suggesting an arrest of their PV biogenesis at the stage of the late endosome . Trypanosoma cruzi invades cells through the endocytic pathway where expression of dominant-negative Rab7 reduces infection, with the same effects found for Rab5 and dynamin . In addition, T. cruzi down-regulates Rab7 in T. cruzi-infected cardiomyocytes . Rab7 may also regulate the maturation of autophagosome-like vacuoles induced by Toxoplasma gondii; experiments inhibiting PI3K, Rab7, vacuolar ATPase and lysosomal enzymes revealed the vacuole/lysosome fusion event mediates antimicrobial activity which is induced by CD40 .
In summary, Rab7 plays a central role in regulating phagosome maturation when cells are infected by microbial pathogens. Microbial pathogens possess survival strategies governed by Rab7, sometimes by employing Rab7 function (e.g. Salmonella) and sometimes by excluding Rab7 function (e.g. Mycobacterium). As shown in Figure 3, microbial pathogens infect cells through phagocytosis and survive in host cells with a similar phagosome-arrest strategy by manipulating the function of Rab proteins. Pathogens enter host cells and reside in nascent phagosomes with reduced acidification. This pathogen-containing phagosome requires Rab5 GTPase to fuse with early endosome to form early phagosome; the early phagosome then fuses with late endosome to form a phagosome, acquiring some late endosomal components, but usually fails to recruit Rab7 GTPase or its partners, preventing phagosome maturation into phagolysosome and allowing pathogen survival in the arrested phagosome. Some pathogen-containing phagosomes are also arrested by partial acquisition of late endosomal components, including Rab7, and form a late endosome/phagosome hybrid, which also cannot mature into a bactericidal phagolysosome. The important roles of Rab7 in diseases caused by micro-organisms suggest that modulation of Rab7 function may be a potential treatment strategy.
Viruses infect cells and take over cellular machineries for replication, viral particle assembly and release. It is well established that membrane trafficking machineries play key roles in the viral life cycle [146–148]. Most enveloped viruses enter cells via clathrin-mediated endocytosis, and are trafficked through the early endodome to the late endosome/lysosome, then release viral materials into cytosol, via an unknown mechanism, due to lysis/leakage of endosomal compartments. It has been revealed that budding and release of some viruses, such as HIV and HBV (hepatitis B virus), requires MVB machineries, such as ESCRT complexes [149–152].
Small GTPases participate in regulating viral life cycle [153,154]. The effects of Rab7 on viral infection were examined for some viruses (Table 3). Miyazawa et al.  reported that internalized Ad7 (adenovirus serotype 7) was co-localized with Rab7 and other late endosomal/lysosomal markers, suggesting that the late endosomal trafficking pathway is involved in viral infection . Sieczkarski and Whittaker  found that dominant-negative Rab7 inhibited infection of influenza virus, without effects on the infection of SFV (Semliki Forest virus) and VSV, which were inhibited by dominant-negative Rab5. However, the findings by Vonderheit and Helenius  indicated that the internalized SFV is finally located to the Rab7 membrane domain, excluding Rab5, Rab4, EEA1 and Arf1, and overexpressing dominant-negative Rab7 resulted in accumulation of SFV in the early endosome . Similarly, Kolokoltsov et al.  found that SFV and VSV entry was reduced by 20% when Rab7-T22N was expressed in cells. Quirin et al.  found SFV infection was not inhibited by the corresponding Rab7-T22N construct, but an SFV mutant stain (SFV fus-1) infection was inhibited by Rab7-T22N. Feng et al.  found that the VSV G protein was accumulated specifically in early endosomes in baby hamster kidney cells expressing the Rab7-N125I mutant. Vidricaire and Tremblay  investigated the roles of Rab7 in HIV-1 infection in polarized human placental cells, and demonstrated that overexpression of both the dominant-negative and dominant-active Rab7 resulted in an almost complete blockade of HIV-1 gene expression (up to 88% inhibition in viral expression was observed). When studying the trafficking of HIV-1 genomic RNA, Lévesque et al.  found that overexpression of RILP had little effect on the synthesis of the polyprotein precursor Pr55Gag, but negatively influenced virus production and infectivity. Recently, van der Schaar et al.  used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking and fusion behaviour of DENV (Dengue virus), and demonstrated that DENV matured in late endosomal compartments by acquisition of Rab7 and loss of Rab5, similar to the phagosome maturation as described above. On the other hand, Krishnan et al.  found that depletion of Rab7 or Rab7 mutant overexpression did not impair DENV and WNV (West Nile virus) infection of HeLa cells. In mammalian cells, data from Kolokoltsov et al.  demonstrated that VEEV (Venezuelan equine encephalitis virus) entry was reduced by Rab7-T22N by 80–90%. Similar findings by Vela et al.  revealed that Pichinde virus enters cells through Rab5–early endosomes, and then uncoats and fuses with Rab7–late endosomes, and knockdown of Rab7 resulted in 80% reduction of viral protein production of Pichinde virus. Ding et al.  showed that overexpression of Rab7 significantly decreased rAAV2 (recombinant adeno-associated virus type-2) transduction. Meertens et al.  found that expression of Rab7-T22N inhibited entry of Ebola virus pseudoparticles. Smith et al.  observed that HPV31 (human papillomavirus type 31) capsids increased residence in the caveosome in mutant-Rab7-transfected cells, but no infection inhibition was detected. The results suggest that, although some discrepancies exist between different studies, Rab7 and Rab7-associated endosomes are intimately involved in entry of some viruses.
Evidence for the involvement of Rab7 in viral infection were also studied in a non-mammalian system. Suppression of PmRab7 (a Rab7 homologue in Penaeus monodon) inhibits the infection of WSSV (white-spot syndrome virus) through interaction with viral protein VP28 , and similar inhibition was also found for YHV (yellow-head virus) in P. monodon . VEEV infection of mosquito cells requires the mosquito homologue of Rab7 . The requirement for Rab7-mediated membrane trafficking in viral infection of a non-mammalian system revealed an evolutionarily conserved pathway.
Rab7 may also be engaged in host-cell defence against viruses; for example, the HIV-1 protein Nef targets MHC-I and CD4 to a Rab7-positive compartment for degradation , which may be the mechanism by which the virus escapes attack from the host's immune system. Taken together, Rab7-mediated endosomal trafficking plays important roles in viral infection; however, the roles of Rab7 in the transport, assembly of newly synthesized viral protein and release of a new viral particle remain unclear. A recent finding by Shah et al.  characterized the group C adenovirus protein RIDα (receptor internalization and degradation α), which interacts with RILP and ORP1L, both of which are effectors of Rab7. The results of this study  also indicated that RIDα mimics the function of Rab7 to recruit RILP to endosomes to facilitate down-regulation of the surface receptor. These data provide another virus–host interaction model. Further studies on RIDα–ORP1L–RILP–Rab7 orchestration may reveal additional mechanisms for Rab7 to regulate viral life cycle.
In summary, Rab7 is engaged in divergent disease pathogenesis, physiological disorders and infectious diseases. The fundamental mechanisms depend on the crucial role of Rab7 in regulating endocytic membrane trafficking, therefore governing the biogenesis of endocytic compartments (late endosome, lysosome, phagosome, autophagosome and other functionally similar organelles) and linking the trafficking events to cell signalling pathways that influence multiple cellular events. Nevertheless, much work remains to further the understanding of pathogenic mechanisms for regulation of diseases by Rab7, such as how the function of Rab7 is regulated by its different effectors. The significance of the interactions between Rab7 and its effectors in regulating disease pathogenesis has not been elucidated in detail. Furthermore, since most investigations are carried out in vitro using cultured cells, the in vivo system using animal models will advance additional knowledge about the underlying mechanisms of Rab7 (or its partners) in endosomal trafficking and its involvement in the development of various diseases.
This work was supported by the start-up funds for new investigators from Xiamen University, People's Republic of China.
Abbreviations: BCV, Brucella-containing vacuole; BMDC, bone-marrow-derived cell; CMT2B, Charcot–Marie–Tooth syndrome type 2B; DENV, Dengue virus; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, EGF receptor; ER, endoplasmic reticulum; ESCRT, endosomal sorting complex required for transport; FSD, functional secretory domain; GAP, GTPase-activation protein; GDI, GDP dissociation inhibitor; GDF, GDI displacement factor; GEF, guanine-nucleotide-exchange factor; HOPS, homotypic fusion and protein sorting; HPS, Hermanskey–Pudlak syndrome; HSAN, hereditary sensory and autonomic neuropathy; Lamp, lysosome-associated membrane protein; LDL, low-density lipoprotein; MVB, multivesicular body; NGF, nerve growth factor; NPC, Niemann–Pick type C; OSBP, oxysterol-binding protein; ORP1L, OSBP-related protein 1; PI3K, phosphoinositide 3-kinase; PV, parasitophorous vacuole; RabGGT, Rab geranylgeranyl transferase; RB, ruffled border; RBG-3, RabGAP initially supposed to target Rab3; REP, Rab escort protein; RIDα, receptor internalization and degradation α; RILP, Rab7-interacting lysosomal protein; SCV, Salmonella-containing vacuole; SFV, Semliki Forest virus; Sif, Salmonella-induced filament; SLSD, sphingolipid storage disease; SNX1/2, sorting nexin 1/2; TrkA, tropomyosin receptor tyrosine kinase A; TRP, tyrosinase-related protein; UVRAG, UV-irradiation resistance-associated gene product; VEEV, Venezuelan equine encephalitis virus; (h)Vps, (human) vacuolar protein sorting; VSV, vesicular stomatitis virus
- © The Authors Journal compilation © 2009 Biochemical Society