Prenylated Rab Acceptor Research Articles

PRAF2 is an oncogene acting to promote the proliferation and invasion of breast cancer cells.

Prenylated rab acceptor 1 domain family member 2 (PRAF2) acts as an oncogene and is closely related to the occurrence and development of various tumors. The present study aimed to clarify the functional relevance of PRAF2 in the biological behaviors of breast cancer by determining the expression of PRAF2 in breast cancer tissues and the corresponding adjacent tissues. The gene phenotypes of PRAF2 in patients with breast cancer in The Cancer Genome Atlas database were predicted using a cancer data online analysis website: The University of Alabama at Birmingham Cancer Data Analaysis Portal (UALCAN). The mRNA and protein expression of PRAF2 was further examined in 37 pairs of fresh frozen breast cancer tissues and adjacent non-tumor tissues by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. High expression of PRAF2 was verified by RT-qPCR in the breast cancer cell line, MCF-7, and small interfering RNA (siRNA) technology was used to silence PRAF2. In the in vitro cell functional experiment, three groups were used: Negative control (NC) group, siRNA-NC group and siRNA-PRAF2 group. Cell Counting Kit-8 (CCK-8) and colony formation assays were conducted to analyze the effect of downregulation of PRAF2 on the proliferation of breast cancer cells. Transwell invasion and cell scratch assays were performed to examine the effect of downregulation of PRAF2 on the invasion and migration of breast cancer cells. UALCAN analysis results indicated that PRAF2 expression was upregulated in breast cancer compared with normal tissue samples (P<0.001). High expression of PRAF2 in breast cancer was associated with TNM stage and regional lymph node metastasis. RT-qPCR results showed increased mRNA expression of PRAF2 in clinical tissue samples from 37 patients with breast cancer, compared with normal adjacent tissues (P<0.001). Protein expression of PRAF2 was also shown to be higher in the breast cancer MCF-7 cells than in the MDA-MB-231 cells. Western blotting analysis combined with ImageJ software quantification showed that the relative expression of PRAF2 protein was significantly higher in clinical tissue samples from 37 patients with breast cancer (1.9750±0.0103) than that in normal adjacent tissues (0.9818±0.0140) (P<0.001). Western blotting analysis results indicated that transfection with siRNA PRAF2 in MCF-7 cells decreased PRAF2 expression (P<0.001). The results of CCK-8 and colony formation assays revealed that downregulation of PRAF2 expression suppressed the proliferation of MCF-7 cells (P<0.05 and P<0.001, respectively). In addition, Transwell invasion and cell scratch assay results showed that downregulation of PRAF2 expression in MCF-7 cells repressed invasion and migration of cancer cells (P<0.001). Overall, PRAF2 expression was significantly higher in breast cancer tissues than normal adjacent tissues, and was closely related to TNM stage and regional lymph node metastasis in breast cancer. PRAF2 was found to act as an oncogene that is able to promote breast cancer cell proliferation and invasion. Thus, PRAF2 may be a potential prognostic factor in patients with breast cancer and a potential target for the treatment of breast cancer metastasis.

Maize HEAT UP-REGULATED GENE 1 plays vital roles in heat stress tolerance.

Increasing temperature is one of the major threats to maize growth and yield globally. Under heat stress conditions, intracellular protein homeostasis is seriously disturbed, leading to accumulation of abnormally folded proteins, especially in the endoplasmic reticulum (ER). Molecular chaperones are vital players in the renaturation process and in preventing protein aggregation. However, heat stress tolerance-associated chaperones are not well documented in maize. Here, we characterized the biological roles of HEAT UP-REGULATED GENE 1 (ZmHUG1) in maize. ZmHUG1 encodes a heat-inducible holdase-type molecular chaperone localized in the ER. Knockout mutant of ZmHUG1 exhibited remarkably enhanced sensitivity to heat stress. Accordingly, the zmhug1 mutant showed severe ER stress under high temperature. MAIZE PRENYLATED RAB ACCEPTOR 1.C1 (ZmPRA1.C1) was identified as a client of ZmHUG1, and heat-induced aggregation of ZmPRA1.C1 was accelerated in the zmhug1 mutant. Furthermore, the expression of ZmHUG1 was rapidly transactivated by ER stress sensor BASIC LEUCINE ZIPPER DOMAIN 60 (bZIP60) when heat stress occurred. This study reveals a ZmHUG1-based thermo-protective mechanism in maize.

Grape Seed Proanthocyanidins (GSPs) Inhibit the Development of Cutaneous Squamous Cell Carcinoma by Regulating the hsa_circ_0070934/miR-136-5p/PRAF2 Axis.

BackgroundGrape seed proanthocyanidins (GSPs) have been shown to inhibit the progression of many cancers, including cutaneous squamous cell carcinoma (CSCC). Circular RNA (circRNA) is a key regulator for cancer progression. However, it is unclear whether GSPs can mediate the progression of CSCC by regulating circRNA.MethodsQuantitative real-time PCR was conducted to determine the expression of hsa_circ_0070934, microRNA (miR)-136-5p and prenylated Rab acceptor family 2 (PRAF2). MTT assay and colony formation assay were used to assess cell proliferation. Cell cycle process and apoptosis were detected by flow cytometry, and cell migration and invasion were measured by transwell assay. Western blot analysis was utilized to examine protein expression. In addition, dual-luciferase reporter assay and RIP assay were used to evaluate the interaction between miR-136-5p and hsa_circ_0070934 or PRAF2. Subcutaneous xenograft models were constructed to explore the function of GSPs on CSCC tumor growth in vivo.ResultsGSPs could reduce hsa_circ_0070934 expression and inhibit CSCC cell proliferation, cell cycle process, migration, invasion, while promote apoptosis. Overexpressed hsa_circ_0070934 could reverse the suppressive effect of GSPs on CSCC cell progression. MiR-136-5p could be sponged by hsa_circ_0070934, and its overexpression also abolished the positively regulation of hsa_circ_0070934 on the progression of GSPs-induced CSCC cells. PRAF2 was a target of miR-136-5p, and its expression could be decreased by GSPs and increased by hsa_circ_0070934. The inhibitory effect of miR-136-5p on CSCC cell progression could be reversed by PRAF2 overexpression. Additionally, GSPs also could inhibit CSCC tumor growth in vivo.ConclusionOur data showed that GSPs regulated the hsa_circ_0070934/miR-136-5p/PRAF2 axis to restrain CSCC progression.

Di-arginine and FFAT-like motifs retain a subpopulation of PRA1 at ER-mitochondria membrane contact sites.

Prenylated Rab Acceptor 1 (PRA1/Rabac1) is a four-pass transmembrane protein that has been found to localize to the Golgi and promiscuously associate with a diverse array of Rab GTPases. We have previously identified PRA1 to be among the earliest significantly down-regulated genes in the rd1 mouse model of retinitis pigmentosa, a retinal degenerative disease. Here, we show that an endogenous subpopulation of PRA1 resides within the endoplasmic reticulum (ER) at ER-mitochondria membrane contact sites in cultured mammalian cells. We also demonstrate that PRA1 contains two previously unidentified ER retention/retrieval amino acid sequences on its cytosolic N-terminal region: a membrane distal di-arginine motif and a novel membrane proximal FFAT-like motif. Using a truncation construct that lacks complete Golgi targeting information, we show that mutation of either motif leads to an increase in cell surface localization, while mutation of both motifs exhibits an additive effect. We also present evidence that illustrates that N- or C- terminal addition of a tag to full-length PRA1 leads to differential localization to either the Golgi or reticular ER, phenotypes that do not completely mirror endogenous protein localization. The presence of multiple ER retention motifs on the PRA1 N-terminal region further suggests that it has a functional role within the ER.

Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis.

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression. To obtain reliable results with this method, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging model for the study of biological asymmetry, biomineralization, and evolution and development. However, no systematic assessment of qPCR reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from several commonly studied tissues in L. stagnalis as well as across the entire body. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, beta-tubulin, ubiquitin, prenylated rab acceptor protein 1, and a voltage gated potassium channel. These genes exhibited a wide range of expression levels among tissues. The tissue-specific stability of each of the genes was consistent when measured by the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our data indicate that the most stable reference genes vary among the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle). Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we provide suggestions for strong pairs of reference genes for single- and multi-tissue analyses of RT-qPCR data in L. stagnalis.

Prenylated Rab acceptor RABAC1 inhibits anti-apoptotic protein BCL2A1 and induces apoptosis

The B cell lymphoma 2 (BCL2) family of proteins constitutes a critical intracellular checkpoint in the intrinsic apoptosis pathway. Among BCL2 members, the anti-apoptotic protein BCL2A1 mediates the resistance to BCL2 inhibitors and may be considered as a target for anti-cancer therapy. Here, we report that prenylated Rab acceptor 1 (RABAC1 or PRA1) inhibits the anti-apoptotic activity of BCL2A1 and induces apoptosis in AGS gastric cancer cells. Protein interaction of BCL2A1 and RABAC1 was verified by an in-vitro glutathione-S-transferase pull-down assay, immunoprecipitation, and confocal microscopy. When apoptosis was induced by cisplatin, the anti-apoptotic activity of BCL2A1 was blocked by RABAC1 expression. RABAC1 caused caspase-3 activation and decreased cell proliferation, clonogenic cell survival, and cell migration and invasion. We suggest RABAC1 as a potential therapeutic target for BCL2A1-related cancer.

Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis.

Identifying and understanding mechanisms that generate phenotypic diversity is a fundamental goal of evolutionary biology. With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail Cepaea nemoralis (Linnaeus, 1758) has been a model for evolutionary biologists and population geneticists for decades. However, the genetic mechanisms by which C. nemoralis generates this pigmented shell diversity remain unknown. An important first step in investigating this pigmentation pattern is to establish a set of validated reference genes for differential gene expression assays. Here we have evaluated eleven candidate genes for reverse transcription quantitative polymerase chain reaction (qPCR) in C. nemoralis. Five of these were housekeeping genes traditionally employed as qPCR reference genes in other species, while six alternative genes were selected de novo from C. nemoralis transcriptome data based on the stability of their expression levels. We tested all eleven candidates for expression stability in four sub-adult tissues of C. nemoralis: pigmented mantle, unpigmented mantle, head and foot. We find that two commonly employed housekeeping genes (alpha tubulin, glyceraldehyde 3-phosphate dehydrogenase) are unsuitable for use as qPCR reference genes in C. nemoralis. The traditional housekeeping gene UBIquitin on the other hand performed very well. Additionally, an RNA-directed DNA polymerase (RNAP), a Potassium Channel Protein (KCHP) and a Prenylated Rab acceptor protein 1 (PRAP), identified de novo from transcriptomic data, were the most stably expressed genes in different tissue combinations. We also tested expression stability over two seasons and found that, although other genes are more stable within a single season, beta actin (BACT) and elongation factor 1 alpha (EF1α) were the most reliable reference genes across seasons.

PRAF2 expression indicates unfavorable clinical outcome in hepatocellular carcinoma.

IntroductionPrenylated Rab acceptor 1 domain family member 2 (PRAF2), a novel oncogene, has been shown to be essential for the development of several human cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear.Materials and methodsPRAF2 mRNA and protein expressions were examined in fresh tissues by quantitative reverse transcription-polymerase chain reaction and Western blot, respectively, and in 518 paraffin-embedded HCC samples by immunohistochemistry. The correlation of PRAF2 expression and clinical outcomes was determined by the Student’s t-test, Kaplan–Meier test, and multivariate Cox regression analysis. The role of PRAF2 in HCC was investigated by cell viability, colony formation, and migration assays in vitro and with a nude mouse model in vivo.ResultsIn our study, the PRAF2 expression was noticeably increased in HCC tissues at both the mRNA and protein levels compared with that of the nontumorous tissues. Kaplan–Meier analysis indicated that high PRAF2 expression was correlated with worse overall survival in a cohort of 518 patients with HCC. The prognostic implication of PRAF2 was verified by stratified survival analysis. The multivariate Cox regression model revealed PRAF2 as an independent poor prognostic factor for overall survival (hazard ratio = 1.244, 95% CI: 1.039–1.498, P<0.017) in HCC. The in vitro data demonstrated that PRAF2 overexpression markedly enhanced cell viability, colony formation, and cell migration. Moreover, ectopic expression of PRAF2 promoted tumor growth and metastasis in vivo.ConclusionCollectively, we conclude that PRAF2 is increased in HCC and is a novel unfavorable biomarker for prognostic prediction for patients with HCC.

Localization of CHMP2B in postnatal rd1 mouse retina.

Retinitis pigmentosa is the most common form of inherited blindness in humans. A well-studied model of the disease is the rd1 mouse, characterized by a loss of function mutation in the catalytic β subunit of the phosphodiesterase 6 (Pde6) holoenzyme involved in phototransduction within rods and cones. The period of photoreceptor degeneration in the rd1 mouse occurs during postnatal days 10-21. In previous work, only Pde6β and vesicular-trafficking protein Prenylated Rab Acceptor 1 (PRA1) have been found to be consistently downregulated during the first ten days following birth. In a yeast-two-hybrid assay conducted by our lab, PRA1 was shown to interact with Charged Multivesicular Body Protein 2B (CHMP2B), an endosomal sorting protein that has been implicated in several neurodegenerative diseases, such as frontotemporal dementia and amyotrophic lateral sclerosis. We investigated whether CHMP2B is mislocalized in the rd1 mouse. Immunohistochemical labeling of CHMP2B was done in both postnatal wild type and rd1 mouse retinas. Prior to the onset of degeneration, CHMP2B immunolabeling was weaker in rd1 retinas, particularly in the developing photoreceptor synaptic layer, compared to wild type. Furthermore, staining of CHMP2B in wild type photoreceptors peaked at postnatal day 12, while CHMP2B staining in rd1 retinas was diffuse and disorganized. In conclusion, these findings show that proper localization of CHMP2B is disrupted in rd1 photoreceptors. Further studies are needed to investigate possible roles for CHMP2B in endocytic activity that is vital to photoreceptor maintenance, as well as differentiation, and development in mouse photoreceptors.

SlPRA1A/RAB attenuate EIX immune responses via degradation of LeEIX2 pattern recognition receptor

ABSTRACTPattern recognition receptors (PRR) are plasma membrane (PM) proteins that recognize microbe-associated molecular patterns (MAMPs), triggering an immune response. PRR are classified as receptor like kinases (RLKs) or receptor like proteins (RLPs). The PM localization of PRRs, which is crucial for their availability to sense MAMPs, depends on their appropriate trafficking through the endomembrane system. Recently, we have identified SlPRA1A, a prenylated RAB acceptor type-1 (PRA1) from S. lycopersicum, as a regulator of RLP-PRR localization and protein levels. SlPRA1A overexpression strongly decreases RLP-PRR protein levels, particularly those of LeEIX2, redirecting it to the vacuole for degradation. Interestingly, SlPRA1A does not affect RLK-PRRs, indicating its activity to be specific to RLP-PRR systems. As PRA1 proteins stabilize RABs on membranes, promoting RABs activity, we aimed to identify a RAB target of SlPRA1A. Screening of a set of A. thaliana RABs revealed that AtRABA1e is able to mimic SlPRA1A activity. Through live cell imaging, we observed that SlPRA1A enhances AtRABA1e localization on SlPRA1A positive punctuated structures. These results indicate that AtRABA1e is a putative target of SlPRA1, and a co-regulator of LeEIX2 trafficking and degradation.

Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

Plants recognize microbial/pathogen associated molecular patterns (MAMP/PAMP) through pattern recognition receptors (PRRs) triggering an immune response against pathogen progression. MAMP/PAMP triggered immune response requires PRR endocytosis and trafficking for proper deployment. LeEIX2 is a well-known Solanum lycopersicum RLP-PRR, able to recognize and respond to the fungal MAMP/PAMP ethylene-inducing xylanase (EIX), and its function is highly dependent on intracellular trafficking. Identifying protein machinery components regulating LeEIX2 intracellular trafficking is crucial to our understanding of LeEIX2 mediated immune responses. In this work, we identified a novel trafficking protein, SlPRA1A, a predicted regulator of RAB, as an interactor of LeEIX2. Overexpression of SlPRA1A strongly decreases LeEIX2 endosomal localization, as well as LeEIX2 protein levels. Accordingly, the innate immune responses to EIX are markedly reduced by SlPRA1A overexpression, presumably due to a decreased LeEIX2 availability. Studies into the role of SlPRA1A in LeEIX2 trafficking revealed that LeEIX2 localization in multivesicular bodies/late endosomes is augmented by SlPRA1A. Furthermore, inhibiting vacuolar function prevents the LeEIX2 protein level reduction mediated by SlPRA1A, suggesting that SlPRA1A may redirect LeEIX2 trafficking to the vacuole for degradation. Interestingly, SlPRA1A overexpression reduces the amount of several RLP-PRRs, but does not affect the protein level of receptor-like kinase PRRs, suggesting a specific role of SlPRA1A in RLP-PRR trafficking and degradation.

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis (Arabidopsis thaliana) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4, atpra1.f4, was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na+/K+-ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA:AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus.

Functional Characterization of a Gene in Sedum alfredii Hance Resembling Rubber Elongation Factor Endowed with Functions Associated with Cadmium Tolerance.

Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the rubber elongation factor gene designated as SaREFl. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, SaREFl-expressing Arabidopsis plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that SaREFl responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of SaREFl as a bait led to the identification of five possible interacting targets in Sedum alfredii Hance. Among them, a gene annotated as prenylated Rab acceptor 1 (PRA1) domain protein was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants.

Anatomical and ultrastructural study of PRAF2 expression in the mouse central nervous system.

Prenylated Rab acceptor family, member 2 (PRAF2) is a four transmembrane domain protein of 19kDa that is highly expressed in particular areas of mammalian brains. PRAF2 is mostly found in the endoplasmic reticulum (ER) of neurons where it plays the role of gatekeeper for the GB1 subunit of the GABAB receptor, preventing its progression in the biosynthetic pathway in the absence of hetero-dimerization with the GB2 subunit. However, PRAF2 can interact with several receptors and immunofluorescence studies indicate that PRAF2 distribution is larger than the ER, suggesting additional biological functions. Here, we conducted an immuno-cytochemical study of PRAF2 distribution in mouse central nervous system (CNS) at anatomical, cellular and ultra-structural levels. PRAF2 appears widely expressed in various regions of mature CNS, such as the olfactory bulbs, cerebral cortex, amygdala, hippocampus, ventral tegmental area and spinal cord. Consistent with its regulatory role of GABAB receptors, PRAF2 was particularly abundant in brain regions known to express GB1 subunits. However, other brain areas where GB1 is expressed, such as basal ganglia, thalamus and hypothalamus, contain little or no PRAF2. In these areas, GB1 subunits might reach the cell surface of neurons independently of GB2 to exert biological functions distinct from those of GABAB receptors, or be regulated by other gatekeepers. Electron microscopy studies confirmed the localization of PRAF2 in the ER, but identified previously unappreciated localizations, in mitochondria, primary cilia and sub-synaptic region. These data indicate additional modes of GABAB regulation in specific brain areas and new biological functions of PRAF2.

Abstract 3323: PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma

Abstract Receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), mediate extracellular signaling that activates downstream cell proliferation, differentiation, and migration. Through ligand stimulation, clathrin-mediated endocytosis regulates the EGFR signal transduction pathway. Prenylated Rab acceptor 1 domain family, membrane 2 (PRAF2) is a novel 19-kDa protein with a prenylated Rab acceptor 1 (PRA1) motif and four transmembrane-spanning domains. Similar to PRAF1 and PRAF3, PRAF2 belongs to the PRAF family of vesicle transport-associated proteins. Our lab identified PRAF2 as a candidate prognostic marker of neuroblastic tumors. Using RNA interference, we showed that PRAF2 plays an essential role in neuroblastoma tumorigenesis and metastasis. In the present study, we examined the function of PRAF2 in EGFR endocytic trafficking. We generated stable PRAF2-knockdown (SK-shPRAF2) and PRAF2-overexpressing (SK-PRAF2) neuroblastoma SK-N-SH cell lines and stimulated both cell lines with EGF (10 ng/ml) for various time periods in order to determine the role of PRAF2 in EGFR endocytic trafficking. As judged by immunofluorescence microscopy, we detected larger Rab5-positive punctuate structures in PRAF2-overexpressing SK-PRAF2 cells compared to PRAF2-knockdown SK-shPRAF2 cells or control cells, after 15 min EGF stimulation. After EGF treatment, PRAF2 clearly colocalized with Rab5-positive punctae in SK-PRAF2 cells, suggesting that PRAF2 interacts with Rab5, a critical regulator for receptor endocytosis and early endosome fusion. Moreover, using a receptor internalization assay, we observed a delay in the internalization of EGFR in EGF-treated SK-shPRAF2 cells, in contrast to SK-PRAF2 and control cells. This is in support of our immunofluorescence microscopy results in which Rab5-postive punctae distribution is observed mostly along the membrane of the EGF-treated SK-shPRAF2 cells. Notably, the level of phosphorylation of Akt (Ser473) transiently elevated in EGF-treated SK-PRAF2 cells after 15 min and diminished thereafter, suggesting that overexpression of PRAF2 accelerates the EGFR internalization and degradation. Based on our findings, we propose that PRAF2 affects EGFR endocytosis by regulating early endosome trafficking and Akt activation in neuroblastoma. Citation Format: Lisette P. Yco, Andre S. Bachmann. PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2014-3323

PRAF2 stimulates cell proliferation and migration and predicts poor prognosis in neuroblastoma

Prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is a novel 19-kDa protein with four transmembrane-spanning domains that belongs to the PRAF protein family. Neuroblastoma (NB) is the most common malignant extracranial solid tumor of childhood that originates in primitive cells of the developing sympathetic nervous system. We investigated the correlation of PRAF2 mRNA expression to NB clinical and genetic parameters using Affymetrix expression analysis of a series of 88 NB tumors and examined the functional role of PRAF2 in an NB cell line using RNA interference. We found that high PRAF2 expression is significantly correlated to several unfavorable NB characteristics: MYCN amplification, high age at diagnosis, poor outcome and high INSS stage. The shRNA-mediated PRAF2 downregulation in the SK-N-SH NB cell line resulted in decreased cellular proliferation, migration and matrix-attachment. These findings were confirmed in NB patient tumor samples, where high PRAF2 expression is significantly correlated to bone and bone marrow metastasis, the main cause of death in NB patients. The present study shows that PRAF2 plays an essential role in NB tumorigenesis and metastasis.

A role for prenylated rab acceptor 1 in vertebrate photoreceptor development

BackgroundThe rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P) 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration.ResultsUsing a microarray approach, we performed gene profiling comparing rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking.ConclusionsWe have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.

NDRG2 and PRA1 interact and synergistically inhibit T-cell factor/β-catenin signaling

NDRG2 is a member of the N-myc downstream regulated gene (NDRG) family, implicated in cell growth and differentiation. Investigation of NDRG2 molecular interactions by yeast two-hybrid screening identified prenylated Rab acceptor-1 (PRA1), involved in vesicle trafficking and protein transport, as binding partner. Binding of NDRG2 (and NDRG1-4) with PRA1 in vitro was confirmed by GST pull-down assay and immunoprecipitation, and colocalization was verified by confocal microscopy in HCT116 cells. Intracellular coexpression showed that NDRG2 and PRA1 synergistically downregulate T-cell factor (TCF) promoter activity and GSK3β phosphorylation. Results suggest that NDRG2 and PRA1 might act synergistically to prevent signaling of TCF/β-catenin. Structured summary of protein interactionsNDRG2abinds to PRA1 by pull down (View interaction)NDRG2aphysically interacts with PRA1 by two hybrid (View Interaction: 1, 2)PRA1physically interacts with NDRG2a by anti tag coimmunoprecipitation (View interaction)NDRG2a, PRA1 and Catenin betacolocalize by cosedimentation (View interaction)NDRG2a and PRA1colocalize by fluorescence microscopy (View Interaction: 1, 2, 3)

The Cytoplasmic Domain of the HIV-1 Glycoprotein gp41 Induces NF-κB Activation through TGF-β-Activated Kinase 1

The human and simian immunodeficiency viruses (HIV and SIV) primarily infect lymphocytes, which must be activated for efficient viral replication. We show that the cytoplasmic domain of the transmembrane glycoprotein gp41 (gp41CD) of both HIV-1 and SIV induces activation of NF-κB, a cellular factor important for proviral genome transcription and lymphocyte activation. This NF-κB activating property localized to a region 12-25 (SIV) or 59-70 (HIV-1) residues from the gp41 membrane-spanning domain. An siRNA-based screen of 42 key NF-κB regulators revealed that gp41CD-mediated activation occurs through the canonical NF-κB pathway via TGF-β-activated kinase 1 (TAK1). TAK1 activity was required for gp41CD-mediated NF-κB activation, and HIV-1-derived gp41CD physically interacted with TAK1 through the same region required for NF-κB activation. Importantly, an NF-κB activation-deficient HIV-1 mutant exhibited increased dependence on cellular activation for replication. These findings demonstrate an evolutionarily conserved role for gp41CD in activating NF-κB to promote infection.

An Arabidopsis Prenylated Rab Acceptor 1 Isoform, AtPRA1.B6, Displays Differential Inhibitory Effects on Anterograde Trafficking of Proteins at the Endoplasmic Reticulum

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aid the targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana) PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to the endoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In this study, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to various endomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat protein complex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescent protein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFP was inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(ΔC):mRFP was not affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency of sporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteins revealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels were controlled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER.