Rab Subfamily Research Articles

Roles of small GTPases in cardiac hypertrophy (Review).

Cardiac hypertrophy results from the heart reacting and adapting to various pathological stimuli and its persistent development is a major contributing factor to heart failure. However, the molecular mechanisms of cardiac hypertrophy remain unclear. Small GTPases in the Ras, Rho, Rab, Arf and Ran subfamilies exhibit GTPase activity and play crucial roles in regulating various cellular responses. Previous studies have shown that Ras, Rho and Rab are closely linked to cardiac hypertrophy and that their overexpression can induce cardiac hypertrophy. Here, we review the functions of small GTPases in cardiac hypertrophy and provide additional insights and references for the prevention and treatment of cardiac hypertrophy.

Abstract 2591: Role of Rab25 in Melanoma and its potential targeting

Abstract Background: RAB25 is a protein that belongs to the RAB11 subfamily located in the lq22 locus of chromosome and is ubiquitously expressed in cells of the gastrointestinal mucosa, ileum, and kidney. Its role in tumorigenesis is variable but is unclear in the case of melanoma progression. Our previous studies showed that loss of RAB25 has a special role in mediating the tumorigenic transformation of human mammary epithelial cells. Its loss particularly cooperated with mutant Ras isoforms to promote transformation in vitro. We are now examining the role of RAB25 in melanomas as melanomas exhibit a high rate of N-Ras mutations. We hypothesize that the presence of NRAS types of mutation and expression of RAB25 are mutually exclusive as RAB25 functions are a tumor suppressor to inhibit this mutation. Methods: Different NRAS mutant melanoma cell lines (SKMEL-2, SKMEL-28, WM1366 and WM266-4) were examined forRab25 expression through qRT-PCR and Western Blotting. The positive control was MCF cell lines and MDA-MB-231 was considered as negative control. Immunohistochemistry was done with biopsy proven melanoma tissue samples and normal adjacent tissue to observe the trend of Rab25 expression. Keratinocytes and fibroblasts in the tissue samples were considered as internal controls.Results: We found a significant decrease in Rab25 expression in NRAS mutant melanoma cell lines compared to Raf mutants and primary melanocytes. The reduction was prominent at both the RNA and protein level. RAB25 expression was also reduced in melanoma tumors as compared to surrounding tissue. Conclusion: RAB25 loss may indeed cooperate with NRAS mutation to promote transformation in melanomas. Further studies including overexpression experiment in the cell line model followed by migration assay, invasion assay, and in vivo nude mouse experiments will provide further confirmation of our hypothesis. Citation Format: Samikshya Kandel, Krishna Rao. Role of Rab25 in Melanoma and its potential targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2591.

A novel membrane targeting domain mediates the endosomal or Golgi localization specificity of small GTPases Rab22 and Rab31

Rab22 and Rab31 belong to the Rab5 subfamily of GTPases that regulates endocytic traffic and endosomal sorting. Rab22 and Rab31 (a.k.a. Rab22b) are closely related and share 87% amino acid sequence similarity, but they show distinct intracellular localization and function in the cell. Rab22 is localized to early endosomes and regulates early endosomal recycling, while Rab31 is mostly localized to the Golgi complex with only a small fraction in the endosomes at steady state. The specific determinants that affect this differential localization, however, are unclear. In this study, we identify a novel membrane targeting domain (MTD) consisting of the C-terminal hypervariable domain (HVD), interswitch loop (ISL), and N-terminal domain as a major determinant of endosomal localization for Rab22 and Rab31, as well as Rab5. Rab22 and Rab31 share the same N-terminal domain, but we find Rab22 chimeras with Rab31 HVD exhibit phenotypic Rab31 localization to the Golgi complex, while Rab31 chimeras with the Rab22 HVD localize to early endosomes, similar to wildtype Rab22. We also find that the Rab22 HVD favors interaction with the early endosomal effector protein Rabenosyn-5, which may stabilize the Rab localization to the endosomes. The importance of effector interaction in endosomal localization is further demonstrated by the disruption of Rab22 endosomal localization in Rabenosyn-5 knockout cells and by the shift of Rab31 to the endosomes in Rabenosyn-5-overexpressing cells. Taken together, we have identified a novel MTD that mediates localization of Rab5 subfamily members to early endosomes via interaction with an effector such as Rabenosyn-5.

Rab22a promotes the proliferation, migration, and invasion of lung adenocarcinoma via up-regulating PI3K/Akt/mTOR signaling pathway

Rab22a, a member of the proto-oncogene RAS family, belongs to the Rab5 subfamily. It participates in early endosome formation and regulates vesicle trafficking. The relationship between Rab22a and tumorigenesis remains elusive. In non-small cell lung cancer specimens, immunohistochemical staining showed consistently high expression of Rab22a in lung adenocarcinoma, but not in squamous cell carcinoma. In lung adenocarcinoma cell lines, A549 and H1299, transfection with Rab22a significantly promoted cell proliferation, migration, and invasion, whereas interference with Rab22a specific siRNA significantly inhibited the above capacities. Transfection with Rab22a also up-regulated the phosphorylation levels of core effector proteins on the PI3K/Akt/mTOR pathway. The Co-IP assay further confirmed the interaction between Rab22a and PI3Kp85α, the core regulatory subunit of PI3K. Application of rapamycin, the mTOR inhibitor, significantly reduced the upregulation of the proliferation, migration, and invasion abilities of lung adenocarcinoma cells transfected with Rab22a. These results suggest that Rab22a can promote the malignant phenotype of lung adenocarcinoma by upregulating the PI3K/Akt/mTOR signaling pathway, and may function as a potential anti-tumor therapeutic target.

Targeted generation of Null Mutants in ZmGDIα confers resistance against maize rough dwarf disease without agronomic penalty.

Targeted generation of Null Mutants in ZmGDIα confers resistance against maize rough dwarf disease without agronomic penalty.

Phylogenetic and Expression Studies of Small GTP-Binding Proteins in Solanum lycopersicum Super Strain B.

This investigation involved a comparative analysis of the small GTPase superfamily in S. lycopersicum super strain B compared to their analogues in leguminous and other non-leguminous species. The small GTPases superfamily members were recognized by tBLASTn searches. The sequences of amino acid were aligned using Clustal Omega and the analysis of phylogeny was performed with the MEGA7 package. Protein alignments were applied for all studied species. Three-dimensional models of RABA2, ROP9, and ROP10 from Solanum lycopersicum “Super strain B” were performed. The levels of mRNA of the Rab, Arf, Rop, and Ran subfamilies were detected in aerial tissues vs. roots. Significant divergences were found in the number of members and groups comprising each subfamily of the small GTPases and Glycine max had the highest count. High expression of Rab and Arf proteins was shown in the roots of legumes whilst in non-legume plants, the highest values were recorded in aerial tissues. S. lycopersicum super strain B had the highest expression of Rab and Arf proteins in its aerial tissues, which may indicate that diazotroph strains have supreme activities in the aerial tissues of strain B and act as associated N-fixing bacteria. The phylogenies of the small GTPase superfamily of the studied plants did not reveal asymmetric evolution of the Ra, Arf, Rop, and Ran subfamilies. Multiple sequence alignments derived from each of the Rab, Arf, and Rop proteins of S. lycopersicum super strain B showed a low frequency of substitutions in their domains. GTPases superfamily members have definite functions during infection, delivery, and maintenance of N2-fixing diazotroph but show some alterations in their function among S. lycopersicum super strain B, and other species.

Use of Immunohistochemistry to Determine Expression of Rab5 Subfamily of GTPases in Mature and Developmental Brains.

Rab GTPases are essentially molecular switches. They serve as master regulators in intracellular membrane trafficking from the formation and transport of vesicles at the originating organelle to its fusion to the membrane at the target organelle. Their functions are diversified and each has their specific subcellular location. Their expression may vary significantly in the same cell when the level of protein production is significantly different in different physiologic status. One of the best examples is the transition from fetal to mature status of cells. Expression and localization of Rab GTPases in mature and developing brains have not been well studied. Immunohistochemistry is an efficient way in the detection, semiquantitation, and localization of Rab GTPases in tissue sections. It is inexpensive and fast which allow efficient mass screening of many sections. In this chapter, we describe the immunohistochemical assay protocol for analyzing several Rab protein expressions of the Rab5 subfamily, including Rab5, Rab17, Rab22, and Rab31, in developmental (fetal) and mature human brains.

Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors.

Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes–late endosomes–lysosomes in osteoclasts.

Upregulation of Rab31 is associated with poor prognosis and promotes colorectal carcinoma proliferation via the mTOR/p70S6K/Cyclin D1 signalling pathway

AimsRab31, a Rab5 subfamily member, has emerged as a modulator of membrane trafficking. Our study serves to clarify the role and mechanism of Rab31 in colorectal carcinoma (CRC) pathogenesis. Materials and methodsThe differential expression of Rab31 was examined in paired normal and cancerous colonic tissues by quantitative PCR, western blot and immunochemistry. The prognostic significance of Rab31 was analysed by univariate and multivariate survival analyses. We also investigated the effects of Rab31 on tumour growth in vitro. Key findingsWe observed that Rab31, which is related to histological differentiation in CRC, was markedly overexpressed in CRC cells. Moreover, patients who showed higher Rab31 levels had a shortened survival period relative to those with low Rab31 levels. Rab31 knockdown significantly downregulated cyclin D1, p-mTOR, and p-p70S6K expression. Moreover, the expression of Rab31-induced p-p70S6K was almost inhibited by rapamycin, a well-established inhibitor of mTOR. Similarly, rapamycin also significantly decreased the stimulatory effect of Rab31 on the expression of cyclin D1. SignificanceThese findings suggested that Rab31 enhanced proliferation, promoted cell cycle progression, and inhibited apoptosis of colorectal carcinoma cells through the mTOR pathway.

In Vitro Fluorescence Resonance Energy Transfer-Based Assay Used to Determine the Rab27-Effector-Binding Affinity.

The Rab27 subfamily consists of Rab27a/b isoforms that have similar but not identical functions. Those functions include the regulation of trafficking, docking, and fusion of various lysosome-related organelles and secretory granules; such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Rab27a/b exert their specific and versatile functions by interacting with 11 effector proteins, preferentially in their GTP-bound state. In recent years, a number of studies have identified roles for Rab27 proteins and their effectors in cancer cell invasion and metastasis, immune response, inflammation, and allergic responses. These findings suggest that Rab27-effector protein interaction inhibitors could contribute to the development of effective strategies to treat these diseases. To facilitate inhibitor identification, in this study we developed a fluorescence resonance energy transfer-based protein-protein interaction assay that reports Rab27-effector interactions. Green fluorescent protein (GFP)-mouse (m) synaptotagmin-like protein (Slp)1 and GFP-mSlp2 (N-terminus Rab27-binding domains) recombinant proteins were used as donor fluorophores, whereas mCherry-human (h) Rab27a/b recombinant proteins were used as acceptor fluorophores. The in vitro binding affinity of mSlp2 to Rab27 was found to be higher compared with mSlp1 and was evidenced by the effective concentration 50 value differences (mSlp2-hRab27b = 0.15 μM < mSlp2-hRab27a = 0.2 μM < mSlp1-hRab27a = 0.32 μM < mSlp1-hRab27b = 0.33 μM). The specificity of the assay was assessed using unlabeled rat (r) Rab27a and hRab27b recombinant proteins as typical competitive inhibitors for Rab27-effector interactions and was evidenced by the inhibitory concentration 50 value differences. Accordingly, this in vitro assay can be employed in identification of candidate inhibitors of Rab27-effector interactions.

Circulating Exosomes from Human Lung Transplant Recipients with Rejection Induces IL-6 by Rab27A Signaling and Promotes Epithelial Mesenchymal Transition of Airway Epithelial Cells In Vitro

Exosomes are small membrane vesicles that regulate intracellular function and cell-to-cell communication. Our report has shown circulating exosomes are induced during lung allograft rejection (Am J Transplant, 2017; 17:474-484). Rab27, a member of the Rab subfamily of GTPases, is essential for exosome secretion. The aim of this study is to determine the role of Rab27A signaling in human bronchial epithelial cells (BEAS-2B) stimulated with exosomes from human lung transplant recipients (LTxRs) diagnosed with acute rejection, bronchiolitis obliterans syndrome (BOS), or stable. Exosomes were isolated using ultracentrifugation and 100µg of exosomes were added to BEAS-2B and incubated for 24hrs. Supernatants collected were analyzed for IL-6 using luminex platform. Epithelial mesenchymal transformation was determined using vimentin and α-SMA by Western blot. Protein band in western blot was normalized with β-Actin and densitometry was performed using Image-J software. Supernatants of BEAS-2B cells, stimulated with BOS-exosome, showed significantly higher production of IL-6 (2927, 4592 MFI p=<0.01) as compared to stable-exosome. Rab27A siRNA knockdown decreased the production of IL-6 (2998 MFI vs 1307 MFI p=<0.01) in supernatants. Further, Rab27A knockdown significantly reduced fibronectin protein in BOS (50% reduction) exosome treated BEAS-2B cells compared to stable (p=<0.01). Vimentin and α-SMA were significantly higher (p=<0.01) in BOS-exosome treated BEAS-2B cells compared to stable-exosome. Rab27A signaling plays an important role in the production of pro-inflammatory cytokine IL-6 by airway epithelial cells following incubation with exosomes isolated from LTxRs with rejection. In addition, Rab27A signaling plays a role in exosome mediated upregulation of fibronectin protein production resulting in epithelia mesenchymal transition of the airway epithelial cells.

Targeting the Small GTPase Superfamily through Their Regulatory Proteins.

The Ras superfamily of small GTPases are guanine‐nucleotide‐dependent switches essential for numerous cellular processes. Mutations or dysregulation of these proteins are associated with many diseases, but unsuccessful attempts to target the small GTPases directly have resulted in them being classed as “undruggable”. The GTP‐dependent signaling of these proteins is controlled by their regulators; guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in the Rho and Rab subfamilies, guanine nucleotide dissociation inhibitors (GDIs). This review covers the recent small molecule and biologics strategies to target the small GTPases through their regulators. It seeks to critically re‐evaluate recent chemical biology practice, such as the presence of PAINs motifs and the cell‐based readout using compounds that are weakly potent or of unknown specificity. It highlights the vast scope of potential approaches for targeting the small GTPases in the future through their regulatory proteins.

Regulation of Small GTPase Prenylation in the Nervous System.

Mevalonate pathway inhibitors have been extensively studied for their roles in cholesterol depletion and for inhibiting the prenylation and activation of various proteins. Inhibition of protein prenylation has potential therapeutic uses against neurological disorders, like neural cancers, neurodegeneration, and neurotramatic lesions. Protection against neurodegeneration and promotion of neuronal regeneration is regulated in large part by Ras superfamily small guanosine triphosphatases (GTPases), particularly the Ras, Rho, and Rab subfamilies. These proteins are prenylated to target them to cellular membranes. Prenylation can be specifically inhibited through altering the function of enzymes of the mevalonate pathway necessary for isoprenoid production and attachment to target proteins to elicit a variety of effects on neural cells. However, this approach does not address how prenylation affects a specific protein. This review focuses on the regulation of small GTPase prenylation, the different techniques to inhibit prenylation, and how this inhibition has affected neural cell processes.

Structural dynamics and allostery of Rab proteins: strategies for drug discovery and design.

Rab proteins represent the largest family of the Rab superfamily guanosine triphosphatase (GTPase). Aberrant human Rab proteins are associated with multiple diseases, including cancers and neurological disorders. Rab subfamily members display subtle conformational variations that render specificity in their physiological functions and can be targeted for subfamily-specific drug design. However, drug discovery efforts have not focused much on targeting Rab allosteric non-nucleotide binding sites which are subjected to less evolutionary pressures to be conserved, hence are likely to offer subfamily specificity and may be less prone to undesirable off-target interactions and side effects. To discover druggable allosteric binding sites, Rab structural dynamics need to be first incorporated using multiple experimentally and computationally obtained structures. The high-dimensional structural data may necessitate feature extraction methods to identify manageable representative structures for subsequent analyses. We have detailed state-of-the-art computational methods to (i) identify binding sites using data on sequence, shape, energy, etc., (ii) determine the allosteric nature of these binding sites based on structural ensembles, residue networks and correlated motions and (iii) identify small molecule binders through structure- and ligand-based virtual screening. To benefit future studies for targeting Rab allosteric sites, we herein detail a refined workflow comprising multiple available computational methods, which have been successfully used alone or in combinations. This workflow is also applicable for drug discovery efforts targeting other medically important proteins. Depending on the structural dynamics of proteins of interest, researchers can select suitable strategies for allosteric drug discovery and design, from the resources of computational methods and tools enlisted in the workflow.

Water in Ras Superfamily Evolution.

The Ras GTPase superfamily of proteins coordinates a diverse set of cellular outcomes, including cell morphology, vesicle transport, and cell proliferation. Primary amino acid sequence analysis has identified Specificity determinant positions (SDPs) that drive diversified functions specific to the Ras, Rho, Rab, and Arf subfamilies (Rojas et al. 2012, J Cell Biol 196:189-201). The inclusion of water molecules in structural and functional adaptation is likely to be a major response to the selection pressures that drive evolution, yet hydration patterns are not included in phylogenetic analysis. This article shows that conserved crystallographic water molecules coevolved with SDP residues in the differentiation of proteins within the Ras superfamily of small GTPases. The patterns of water conservation between protein subfamilies parallel those of sequence-based evolutionary trees. Thus, hydration patterns have the potential to help elucidate functional significance in the evolution of amino acid residues observed in phylogenetic analysis of homologous proteins. © 2019 Wiley Periodicals, Inc.

Comprehensive knockout analysis of the Rab family GTPases in epithelial cells.

The Rab family of small GTPases comprises the largest number of proteins (∼60 in mammals) among the regulators of intracellular membrane trafficking, but the precise function of many Rabs and the functional redundancy and diversity of Rabs remain largely unknown. Here, we generated a comprehensive collection of knockout (KO) MDCK cells for the entire Rab family. We knocked out closely related paralogs simultaneously (Rab subfamily knockout) to circumvent functional compensation and found that Rab1A/B and Rab5A/B/C are critical for cell survival and/or growth. In addition, we demonstrated that Rab6-KO cells lack the basement membrane, likely because of the inability to secrete extracellular matrix components. Further analysis revealed the general requirement of Rab6 for secretion of soluble cargos. Transport of transmembrane cargos to the plasma membrane was also significantly delayed in Rab6-KO cells, but the phenotype was relatively mild. Our Rab-KO collection, which shares the same background, would be a valuable resource for analyzing a variety of membrane trafficking events.

Structural basis of guanine nucleotide exchange for Rab11 by SH3BP5.

The Rab GTPase family is a major regulator of membrane traffic in eukaryotic cells. The Rab11 subfamily plays important roles in specific trafficking events such as exocytosis, endosomal recycling, and cytokinesis. SH3BP5 and SH3BP5-like (SH3BP5L) proteins have recently been found to serve as guanine nucleotide exchange factors (GEF) for Rab11. Here, we report the crystal structures of the SH3BP5 GEF domain alone and its complex with Rab11a. SH3BP5 exhibits a V-shaped structure comprising two coiled coils. The coiled coil composed of α1, and α4 is solely responsible for the Rab11a binding and GEF activity. SH3BP5 pulls out and deforms switch I of Rab11a so as to facilitate the GDP release from Rab11a. SH3BP5 interacts with the N-terminal region, switch I, interswitch, and switch II of Rab11a. SH3BP5 and SH3BP5L localize to Rab11-positive recycling endosomes and show GEF activity for all of the Rab11 family but not for Rab14. Fluorescence-based GEF assays combined with site-directed mutagenesis reveal the essential interactions between SH3BP5 and Rab11 family proteins for the GEF reaction on recycling endosomes.

RAL GTPases: Biology and Potential as Therapeutic Targets in Cancer.

More than a hundred proteins comprise the RAS superfamily of small GTPases. This family can be divided into RAS, RHO, RAB, RAN, ARF, and RAD subfamilies, with each shown to play distinct roles in human cells in both health and disease. The RAS subfamily has a well-established role in human cancer with the three genes, HRAS, KRAS, and NRAS being the commonly mutated in tumors. These RAS mutations, most often functionally activating, are especially common in pancreatic, lung, and colorectal cancers. Efforts to inhibit RAS and related GTPases have produced inhibitors targeting the downstream effectors of RAS signaling, including inhibitors of the RAF-mitogen-activated protein kinase/extracellular signal-related kinase (ERK)-ERK kinase pathway and the phosphoinositide-3-kinase-AKT-mTOR kinase pathway. A third effector arm of RAS signaling, mediated by RAL (RAS like) has emerged in recent years as a critical driver of RAS oncogenic signaling and has not been targeted until recently. RAL belongs to the RAS branch of the RAS superfamily and shares a high structural similarity with RAS. In human cells, there are two genes, RALA and RALB, both of which have been shown to play roles in the proliferation, survival, and metastasis of a variety of human cancers, including lung, colon, pancreatic, prostate, skin, and bladder cancers. In this review, we summarize the latest knowledge of RAL in the context of human cancer and the recent advancements in the development of cancer therapeutics targeting RAL small GTPases.

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation.

PurposeThe serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression.Materials and MethodsCEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis.ResultsCEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner.ConclusionCEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.