Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.     

Fasciculation and elongation protein zeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrial motility

The fasciculation and elongation protein zeta-1 (FEZ1), a mammalian orthologue of Caenorhabditis elegans UNC-76 protein, is a 45-kDa protein with four coiled-coiled domains and efficiently promotes the neurite elongation in the rat phaeochromocytoma PC12 cells. UNC-76 proteins of C. elegans and Drosophila have been genetically demonstrated to be involved in the axonal guidance. We here show that FEZ1 RNA interference (RNAi) represses the formation of axon in rat embryo hippocampal neurons. An anterograde mitochondrial movement is also retarded in neurites of the RNAi-treated hippocampal neurons. Moreover, the size of mitochondria is considerably elongated by the RNAi treatment. The transport of mitochondria from soma to axon or dendrites is essential for the neuronal differentiation. Therefore, our results strongly suggest that FEZ1 participates in the establishment of neuronal polarity by controlling the mitochondrial motility along axon.     

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain

Background

Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.

Methodology/Principal Findings

In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.

Conclusions/Significance

Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.     

Fasciculation and elongation zeta proteins 1 and 2: From structural flexibility to functional diversity

Fasciculation and elongation zeta/zygin(FEZ) proteins are a family of hub proteins and share many characteristics like high connectivity in interaction networks, they are involved in several cellular processes, evolve slowly and in general have intrinsically disordered regions. In 1985, unc-76 gene was firstly described and involved in axonal growth in C. elegans, and in 1997 Bloom and Horvitz enrolled also the human homologues genes, FEZ1 and FEZ2, in this process. While nematodes possess one gene(unc-76), mammalians have one more copy(FEZ1 and FEZ2). Several animal models have been used to study FEZ family functions like: C. elegans, D. melanogaster, R. novergicus and human cells.Complementation assays were performed and demonstrated the function conservation between paralogues. Human FEZ1 protein is more studied followed by UNC-76 and FEZ2 proteins, respectively. While FEZ1 and UNC-76 shared interaction partners, FEZ2 evolved and increased the number of protein-protein interactions(PPI) with cytoplasmatic partners. FEZ proteins are implicated in intracellular transport, acting as bivalent cargo transport adaptors in kinesinmediated movement. Especially in light of this cellular function, this family of proteins has been involved in several processes like neuronal development,neurological disorders, viral infection and autophagy. However, nuclear functions of FEZ proteins have been explored as well, due to high content of PPI with nuclear proteins, correlating FEZ1 expression to Sox2 and Hoxb4 gene regulation and retinoic acid signaling. These recent findings open new avenue to study FEZ proteins functions and its involvement in already described processes.This review intends to reunite aspects of evolution, structure, interaction partners and function of FEZ proteins and correlate them to physiological and pathological processes.     

Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH₂-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.     

Identification of FEZ1 as a protein that interacts with JC virus agnoprotein and microtubules: role of agnoprotein-induced dissociation of FEZ1 from microtubules in viral propagation

The human polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy, and encodes six major proteins, including agnoprotein. Agnoprotein colocalizes with microtubules in JCV-infected cells, but its function is not fully understood. We have now identified fasciculation and elongation protein zeta 1 (FEZ1) as a protein that interacted with JCV agnoprotein in a yeast two-hybrid screen of a human brain cDNA library. An in vitro binding assay showed that agnoprotein interacted directly with FEZ1 and microtubules. A microtubule cosedimentation assay revealed that FEZ1 also associates with microtubules and that agnoprotein induces the dissociation of FEZ1 from microtubules. Agnoprotein inhibited the promotion by FEZ1 of neurite outgrowth in PC12 cells. Conversely, overexpression of FEZ1 suppressed JCV protein expression and intracellular trafficking in JCV-infected cells. These results suggest that FEZ1 promotes neurite extension through its interaction with microtubules, and that agnoprotein facilitates JCV propagation by inducing the dissociation of FEZ1 from microtubules.     

Identification of a tissue-non-specific homologue of axonal fasciculation and elongation protein zeta-1

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein involved in the axonal outgrowth and fasciculation and promotes neurite extension of PC12 cells through interaction with protein kinase C zeta (PKCzeta). The gene coding for FEZ2, a homologue of FEZ1, has also been reported in rat and human. In this study, we compared mRNA expression of FEZ1 and FEZ2 in adult rat tissues and mouse embryos by Northern blot and in situ hybridization analyses. In contrast to FEZ1 whose mRNA is expressed almost exclusively in rat brain and temporarily around the neurogenesis stage of mouse embryos, the message for FEZ2 is detected weakly in most tissues and abundantly throughout the mouse embryonic stages. Similar to FEZ1, FEZ2 interacted with PKCzeta and induced neurite extension of PC12 cells when coexpressed with a constitutively active mutant of PKCzeta. These results suggest that FEZ2 plays an important role in the morphological changes of various cells by associating with PKCzeta in a tissue-non-specific manner.     

Spindle Assembly Checkpoint Protein Dynamics Reveal Conserved and Unsuspected Roles in Plant Cell Division

Background

In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle.

Methodology/Principal Findings

We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of ‘wait anaphase’, plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants.

Conclusions/Significance

We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division.     

Expression of fasciculation and elongation protein zeta-1 (FEZ1) in cultured rat neonatal astrocytes

Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear. A DNA microarray was used to identify gene expression in cultured T1As and T2As isolated from postnatal day 1 rat cortex. Ninety-nine of the 138 differentially expressed genes were involved in a diverse number of processes. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes. RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1 and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus. The results contribute to a clearer understanding of the two types of astrocytes.     

Downregulation of Keratin 76 Expression during Oral Carcinogenesis of Human,Hamster and Mouse

Background

Keratins are structural marker proteins with tissue specific expression; however, recent reports indicate their involvement in cancer progression. Previous study from our lab revealed deregulation of many genes related to structural molecular integrity including KRT76. Here we evaluate the role of KRT76 downregulation in oral precancer and cancer development.

Methods

We evaluated KRT76 expression by qRT-PCR in normal and tumor tissues of the oral cavity. We also analyzed K76 expression by immunohistochemistry in normal, oral precancerous lesion (OPL), oral squamous cell carcinoma (OSCC) and in hamster model of oral carcinogenesis. Further, functional implication of KRT76 loss was confirmed using KRT76-knockout (KO) mice.

Results

We observed a strong association of reduced K76 expression with increased risk of OPL and OSCC development. The buccal epithelium of DMBA treated hamsters showed a similar trend. Oral cavity of KRT76-KO mice showed preneoplastic changes in the gingivobuccal epithelium while no pathological changes were observed in KRT76 negative tissues such as tongue.

Conclusion

The present study demonstrates loss of KRT76 in oral carcinogenesis. The KRT76-KO mice data underlines the potential of KRT76 being an early event although this loss is not sufficient to drive the development of oral cancers. Thus, future studies to investigate the contributing role of KRT76 in light of other tumor driving events are warranted.     

The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system

Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.     

UNC-52/perlecan affects gonadal leader cell migrations in C. elegans hermaphrodites through alterations in growth factor signaling

The unc-52 gene of Claenorhabditis elegans encodes a homologue of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. We propose that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways.     

Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of β-amyloid plaques in the brain. Plaques are composed of the amyloid-β peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer''s Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality.

Methodology/Principal Findings

We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads to the retention of APL-1 in the cell body.

Conclusions/Significance

Our results reveal novel insights into the intracellular trafficking of APL-1 and we report a functional role for APL-1 in synaptic transmission.     

RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer

Purpose

Pancreatic cancer is an aggressive malignancy with characteristic metastatic course of disease and resistance to conventional chemo-radiotherapy. RLIP76 is a multi-functional cell membrane protein that functions as a major mercapturic acid pathway transporter as well as key regulator of receptor-ligand complexes. In this regard, we investigated the significance of targeting RLIP76 on PI3K/Akt pathway and mechanisms regulating response to chemo-radiotherapy.

Research Design and Methods

Cell survival was assessed by MTT and colony forming assays. Cellular levels of proteins and phosphorylation was determined by Western blot analyses. The impact on apoptosis was determined by TUNEL assay. The anti-cancer effects of RLIP76 targeted interventions in vivo were determined using mice xenograft model of the pancreatic cancer. The regulation of doxorubicin transport and radiation sensitivity were determined by transport studies and colony forming assays, respectively.

Results

Our current studies reveal an encompassing model for the role of RLIP76 in regulating the levels of fundamental proteins like PI3K, Akt, E-cadherin, CDK4, Bcl2 and PCNA which are of specific importance in the signal transduction from critical upstream signaling cascades that determine the proliferation, apoptosis and differentiation of pancreatic cancer cells. RLIP76 depletion also caused marked and sustained regression of established human BxPC-3 pancreatic cancer tumors in nude mouse xenograft model. RLIP76 turned out to be a major regulator of drug transport along with contributing to the radiation resistance in pancreatic cancer.

Conclusions/Significance

RLIP76 represents a mechanistically significant target for developing effective interventions in aggressive and refractory pancreatic cancers.     

CSN-5, a Component of the COP9 Signalosome Complex,Regulates the Levels of UNC-96 and UNC-98, Two Components of M-lines in Caenorhabditis elegans Muscle

In Caenorhabditis elegans two M-line proteins, UNC-98 and UNC-96, are involved in myofibril assembly and/or maintenance, especially myosin thick filaments. We found that CSN-5, a component of the COP9 signalosome complex, binds to UNC-98 and -96 using the yeast two-hybrid method. These interactions were confirmed by biochemical methods. The CSN-5 protein contains a Mov34 domain. Although one other COP9 signalosome component, CSN-6, also has a Mov34 domain, CSN-6 did not interact with UNC-98 or -96. Anti-CSN-5 antibody colocalized with paramyosin at A-bands in wild type and colocalized with abnormal accumulations of paramyosin found in unc-98, -96, and -15 (encodes paramyosin) mutants. Double knockdown of csn-5 and -6 could slightly suppress the unc-96 mutant phenotype. In the double knockdown of csn-5 and -6, the levels of UNC-98 protein were increased and the levels of UNC-96 protein levels were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Taken together, we found that CSN-5 functions in muscle cells to regulate UNC-98 and -96, two M-line proteins.     

Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.