Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two-hybrid system

The TPD52 (tumor protein D52)-like proteins are small coiled-coil motif-bearing proteins which were first identified though their expression in human breast carcinoma. TPD52-like proteins are known to interact in hetero-and homomeric fashions, but there are no known heterologous binding partners for these proteins. We now report the cloning of a novel member of the MAL proteolipid family, named MAL2, though its interaction with a TPD52L2 bait in a yeast two-hybrid screen. MAL2 is predicted to be 176 residues (19 kDa) with four transmembrane domains and is 35.8% identical to MAL, a proteolipid required in apical vesicle transport. The MAL2 prey bound all TPD52-like baits tested in the yeast two-hybrid system and in vitro translation of MAL2 produced a single 19-kDa (35)S-labeled protein which specifically bound full-length GST-Tpd52 in GST pull-down assays. The gene MAL2, which was localized to human chromosomal band 8q23 and shown to consist of four exons, is predominantly expressed in human kidney, lung, and liver. Our study has therefore identified a novel member of the MAL proteolipid family and potentially implicates TPD52-like proteins in vesicle transport.     

hABCF3, a TPD52L2 interacting partner, enhances the proliferation of human liver cancer cell lines in vitro

In the present study, we characterized an evolutionarily conserved non-transmembrane ATP-binding cassette protein: hABCF3. Subcellular immunofluorescence staining demonstrated that hABCF3 localizes preferentially in cytoplasm, unlike its paralog protein hABCF1, which localizes in both cytoplasm and nucleus. Quantitative realtime PCR analysis revealed that hABCF3 is expressed in all tissues examined, with high expression level in heart, liver, and pancreas. Interestingly, ectopic hABCF3 promoted proliferation of human liver cancer cell lines. Moreover, knock down of hABCF3 protein expression by siRNA inhibited cell proliferation. In addition, we identified TPD52L2 (Tumor Protein D52-like 2) as a hABCF3 interacting protein via yeast two-hybrid. This interaction was further confirmed by in vivo co-immunoprecipitation and co-localization assays. Furthermore, we identified the interactional region of hABCF3 to be the first 200 amino acids uncharacterized region. Notably, the truncated version of hABCF3, which lacks the TPD52L2 binding region, remarkably impaired hABCF3-mediated cell proliferation. Taken together, these findings suggest that hABCF3 positively regulates cell proliferation, at least partially through the interaction with a tumor protein D52 protein family member: TPD52L2.     

A testis-specific and testis developmentally regulated tumor protein D52 (TPD52)-like protein TPD52L3/hD55 interacts with TPD52 family proteins

Tumor protein D52-like proteins (TPD52) are small coiled-coil motif bearing proteins that were first identified in breast cancer. TPD52 and related proteins have been implicated in cell proliferation, apoptosis, and vesicle trafficking. To date, three human TPD52 members had been identified, named hD52 (TPD52), hD53 (TPD52L1), and hD54 (TPD52L2). The most important characteristic of the protein family is a highly conserved coiled-coil motif that is required for homo- and heteromeric interaction with other TPD52-like proteins. Herein, we identified a novel TPD52-like sequence (TPD52L3, or hD55) in human testis using cDNA microarray. Sequence analysis of the deduced protein suggests that hD55 contains a coiled-coil motif and is highly conserved compared with other TPD52-like sequences. Yeast two-hybrid and GST pull-down assays revealed that hD55 interacts with hD52, hD53, hD54, and itself. cDNA microarray detection found that hD55 was expressed at 5.6-fold higher levels in adult testis than in fetal testis. Additionally, the expression profile shows that hD55 is testis-specific, indicating a potential role for hD55 in testis development and spermatogenesis.     

PLP2/A4 interacts with CCR1 and stimulates migration of CCR1-expressing HOS cells

Multiple CC chemokines bind to CCR1, which plays important roles in immune and inflammatory responses. To search for proteins involved in the CCR1 signaling pathway, we screened a yeast two-hybrid library using the cytoplasmic tail of CCR1 as the bait. One of the positive clones contained an open reading frame of 456bp, of which the nucleotide sequence was identical to that of proteolipid protein 2 (PLP2), also known as protein A4. Mammalian two-hybrid and coimmunoprecipitation analyses demonstrated the association of PLP2/A4 with CCR1. Indirect immunofluorescence analysis revealed that PLP2/A4 was predominantly located in plasma membrane and colocalized with CCR1 in transfected human HEK293 cells. In addition, focal staining of CCR1 appeared on the periphery of the membrane upon short exposure to Leukotactin-1(Lkn-1)/CCL15, a CCR1 agonist, and was costained with PLP2/A4 on the focal regions. PLP2/A4 mRNAs were detected in various cells such as U-937, HL-60, HEK293, and HOS cells. Overexpression of PLP2/A4 stimulated a twofold increase in the agonist-induced migration of HOS/CCR1 cells, implicating a functional role for PLP2/A4 in the chemotactic processes via CCR1.     

The role of the coiled-coil motif in interactions mediated by TPD52

TPD52 (D52)-like proteins are small coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma that mutually interact in hetero- and homomeric fashions. However, it has been unclear whether the coiled-coil motif is sufficient, or even necessary, for these interactions to occur. We have therefore examined the binding activities of a panel of C-terminally deleted D52 proteins in both the yeast two-hybrid system and pull-down assays. In the yeast two-hybrid system, interactions were only detected when regions C-terminal to the coiled-coil motif were also present. However, using pull-down assays, interactions were detected for all deletion mutants which included the coiled-coil motif. This suggests that the coiled-coil motif is indeed necessary for interactions mediated by D52 proteins, but that C-terminal protein regions facilitate and/or stabilize these interactions.     

Membrane targeting by APPL1 and APPL2: dynamic scaffolds that oligomerize and bind phosphoinositides

Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane-targeting properties of the APPL1 and APPL2 Bin/Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunoprecipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding.     

Plant RABs: Role in Development and in Abiotic and Biotic Stress Responses

Endosomal trafficking plays an integral role in various eukaryotic cellular activities and is vital for higher-order functions in multicellular organisms. RAB GTPases are important proteins that influence various aspects of membrane traffic, which consequently influence many cellular functions and responses. Compared to yeast and mammals, plants have evolved a unique set of plant-specific RABs that play a significant role in their development. RABs form the largest family of small guanosine triphosphate (GTP)-binding proteins, and are divided into eight sub-families named RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Recent studies on different species suggest that RAB proteins play crucial roles in intracellular trafficking and cytokinesis, in autophagy, plant microbe interactions and in biotic and abiotic stress responses. This review recaptures and summarizes the roles of RABs in plant cell functions and in enhancing plant survival under stress conditions.     

采用酵母双杂交法筛选HbMyb1相互作用蛋白

HbMyb1是橡胶树死皮病相关基因,为了进一步研究HbMyb1的功能,采用酵母双杂交方法,从胶乳cDNA文库中筛选出10个与HbMyb1发生相互作用的蛋白,包括已知的橡胶延伸因子和橡胶小粒子蛋白等,其中4个分别与3-羟酰基-ACP-脱氢酶、NAD-依赖的山梨糖醇脱氢酶、氧化还原酶、假定的富含亮氨酸重复蛋白具有同源性的蛋白,其余4个为未知蛋白。     

Tumor protein D52 (isoform 3) interacts with and promotes peroxidase activity of Peroxiredoxin 1 in prostate cancer cells implicated in cell growth and migration

Tumor protein D52 (TPD52) is overexpressed in multiple cancers including prostate cancer due to gene amplification and investigations to understand its role in the pathophysiology of different cancers are continuing. GST pull-down assays and Tandem affinity purification of TPD52 as bait identified novel prey Peroxiredoxin 1 (PRDX1) in prostate cancer (PCa) cells. PRDX1 interaction with TPD52 was confirmed in immunoprecipitation and affinity interaction assays. Mapping of interaction domain indicated that PRDX1 interacts with C-terminal region of TPD52 containing PEST domain between 152 and 179 amino acids, a new binding region of TPD52. Here we show that TPD52 interaction with PRDX1 increased its peroxidase activity and ectopic expression of TPD52 induced dimerization of PRDX1 in PCa cells. Moreover, H₂O₂ exposure evoked the interaction between TPD52 and PRDX1 while depletion of both proteins led to the accumulation of H₂O₂ suggesting peroxidase activity is important to maintain oxidative capacity in PCa cells. We also observed that overexpression or downregulation of TPD52 and PRDX1 individually or together affecting PCa cells growth, survival, and migration. Altogether, our results show a novel interaction partner of TPD52 providing new insights of its functions and ascertain the role of TPD52-PRDX1 interaction in PCa progression.     

PAS/LOV proteins: A proposed new class of plant blue light receptor

The light, oxygen or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor.Key words: PAS, LOV, blue light, protein-protein interaction, photoreceptor     

Intracellular nanovesicles mediate α5β1 integrin trafficking during cell migration

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs.     

The small GTPase RAB37 functions as an organizer for autophagosome biogenesis

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. How autophagosomal vesicle forms in a spatio-temporally regulated manner remains elusive. Our recent study revealed that small GTPase, RAB37 (RAB37, member RAS oncogene family), functions as a key organizer of autophagosomal membrane biogenesis. RAB37 interacts with ATG5 (autophagy related 5) and promotes autophagosome formation by modulating ATG12–ATG5-ATG16L1 complex assembly. These findings provide new insights into autophagy regulation.     

The RAB5‐GEF Function of RIN1 Regulates Multiple Steps During Listeria monocytogenes Infection

Listeria monocytogenes is a food‐borne pathogenic bacterium that invades intestinal epithelial cells through a phagocytic pathway that relies on the activation of host cell RAB5 GTPases. Listeria monocytogenes must subsequently inhibit RAB5, however, in order to escape lysosome‐mediated destruction. Relatively little is known about upstream RAB5 regulators during L. monocytogenes entry and phagosome escape processes in epithelial cells. Here we identify RIN1, a RAS effector and RAB5‐directed guanine nucleotide exchange factor (GEF), as a host cell factor in L. monocytogenes infection. RIN1 is rapidly engaged following L. monocytogenes infection and is required for efficient invasion of intestinal epithelial cells. RIN1‐mediated RAB5 activation later facilitates the fusion of phagosomes with lysosomes, promoting clearance of bacteria from the host cell. These results suggest that RIN1 is a host cell regulator that performs counterbalancing functions during early and late stages of L. monocytogenes infection, ultimately favoring pathogen clearance.      

Classical swine fever virus non-structural protein 4B binds tank-binding kinase 1

Classical swine fever (CSF) is a contagious disease with a high mortality rate and is caused by classical swine fever virus (CSFV). CSFV non-structural protein 4B (NS4B) plays a crucial role in CSFV replication and pathogenicity. However, precisely how NS4B exerts these functions remains unknown, especially as there are no reports relating to potential cellular partners of CSFV NS4B. Here, a yeast two-hybrid (Y2H) system was used to screen the cellular proteins interacting with NS4B from a porcine alveolar macrophage (PAM) cDNA library. The protein screen along with alignment using the NCBI database revealed 14 cellular proteins that interact with NS4B: DDX39B, COX7C, FTH1, MAVS, NR2F6, RPLP1, PSMC4, FGL2, MKRN1, RPL15, RPS3, RAB22A, TP53BP2 and TBK1. These proteins mostly relate to oxidoreductase activity, signal transduction, localization, biological regulation, catalytic activity, transport and metabolism by GO categories. Tank-binding kinase 1 (TBK1) was chosen for further confirmation. The NS4B-TBK1 interaction was further confirmed by subcellular co-location, co-immunoprecipitation and glutathione S-transferase pull-down assays. This study offers a theoretical foundation for further understanding of the diversity of NS4B functions in relation to viral infection and subsequent pathogenesis.     

NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition. However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.     

Identification of mutations in protein kinase CKIIbeta subunit that affect its binding to ribosomal protein L41 and homodimerization

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The CKIIbeta subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of CKIIbeta that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of CKIIbeta are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for CKIIbeta homodimerization. Two point mutants, R75 and R83, of CKIIbeta interacted with L5, topoisomerase IIbeta, and CKBBP1/SAG, but not with the wild-type CKIIbeta. This indicates that CKIIbeta homodimerization is not a prerequisite for its binding to target proteins. These CKIIbeta point mutants may be useful in exploring the biochemical physiological functions of CKIIbeta.     

Chromosome assignment of four RAS-related RAB genes

Summary The human RAB genes share structural and biochemical properties with the RAS gene superfamily. The encoded RAB proteins show 38 to 75% amino acid identity with the yeast YPT1 and SEC4 gene products. We used four human RAB-cDNAs, RAB3B, RAB4, RAB5 and RAB6, to map the corresponding genes on human chromosomes. These genes were assigned to 1p32-p31, 1q42-q43, 3p24-p22 and 2q14-q21, respectively, by in situ hybridization.     

NDP52 SUMOylation contributes to low-dose X-rays-induced cardiac hypertrophy through PINK1/Parkin-mediated mitophagy via MUL1/SUMO2 signalling

Radiation-induced heart damage caused by low-dose X-rays has a significant impact on tumour patients' prognosis, with cardiac hypertrophy being the most severe noncarcinogenic adverse effect. Our previous study demonstrated that mitophagy activation promoted cardiac hypertrophy, but the underlying mechanisms remained unclear. In the present study, PARL-IN-1 enhanced excessive hypertrophy of cardiomyocytes and exacerbated mitochondrial damage. Isobaric tags for relative and absolute quantification-based quantitative proteomics identified NDP52 as a crucial target mediating cardiac hypertrophy induced by low-dose X-rays. SUMOylation proteomics revealed that the SUMO E3 ligase MUL1 facilitated NDP52 SUMOylation through SUMO2. Co-IP coupled with LC–MS/MS identified a critical lysine residue at position 262 of NDP52 as the key site for SUMO2-mediated SUMOylation of NDP52. The point mutation plasmid NDP52^K262R inhibited mitophagy under MUL1 overexpression, as evidenced by inhibition of LC3 interaction with NDP52, PINK1 and LAMP2A. A mitochondrial dissociation study revealed that NDP52^K262R inhibited PINK1 targeting to endosomes early endosomal marker (EEA1), late/lysosome endosomal marker (LAMP2A) and recycling endosomal marker (RAB11), and laser confocal microscopy confirmed that NDP52^K262R impaired the recruitment of mitochondria to the autophagic pathway through EEA1/RAB11 and ATG3, ATG5, ATG16L1 and STX17, but did not affect mitochondrial delivery to lysosomes via LAMP2A for degradation. In conclusion, our findings suggest that MUL1-mediated SUMOylation of NDP52 plays a crucial role in regulating mitophagy in the context of low-dose X-ray-induced cardiac hypertrophy. Two hundred sixty-second lysine of NDP52 is identified as a key SUMOylation site for low-dose X-ray promoting mitophagy activation and cardiac hypertrophy. Collectively, this study provides novel implications for the development of therapeutic strategies aimed at preventing the progression of cardiac hypertrophy induced by low-dose X-rays.