Trans-Golgi Network-An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli.

Varicella-zoster virus ORF7 interacts with ORF53 and plays a role in its trans-Golgi network localization

Varicella-zoster virus(VZV) is a neurotropic alphaherpesvirus that causes chickenpox and shingles. ORF7 is an important virulence determinant of VZV in both human skin and nerve tissues,however, its specific function and involved molecular mechanism in VZV pathogenesis remain largely elusive. Previous yeast two-hybrid studies on intraviral protein-protein interaction network in herpesviruses have revealed that VZV ORF7 may interact with ORF53, which is a virtually unstudied but essential viral protein. The aim of this study is to identify and characterize VZV ORF53, and to investigate its relationship with ORF7. For this purpose, we prepared monoclonal antibodies against ORF53 and, for the first time, characterized it as a ~40 k Da viral protein predominantly localizing to the trans-Golgi network of the infected host cell. Next, we further confirmed the interaction between ORF7 and ORF53 by co-immunoprecipitation and co-localization studies in both plasmid-transfected and VZV-infected cells. Moreover, interestingly, we found that ORF53 lost its trans-Golgi network localization and became dispersed in the cytoplasm of host cells infected with an ORF7-deleted recombinant VZV, and thus ORF7 seems to play a role in normal subcellular localization of ORF53. Collectively, these results suggested that ORF7 and ORF53 may function as a complex during infection, which may be implicated in VZV pathogenesis.

Two NPF transporters mediate iron long-distance transport and homeostasis in Arabidopsis

Iron(Fe)transport and reallocation are essential to Fe homeostasis in plants,but it is unclear how Fe homeostasis is regulated,especially under stress.Here we report that NPF5.9 and its close homolog NPF5.8 redundantly regulate Fe transport and reallocation in Arabidopsis.NPF5.9 is highly upregulated in response to Fe deficiency.NPF5.9 expresses preferentially in vasculature tissues and localizes to the trans-Golgi network,and NPF5.8 showed a similar expression pattern.Long-distance Fe transport and allocation into aerial parts was significantly increased in NPF5.9-overexpressing lines.In the double mutant npf5.8 npf5.9,Fe loading in aerial parts and plant growth were decreased,which were partially rescued by Fe supplementation.Further analysis showed that expression of PYE,the negative regulator for Fe homeostasis,and its downstream target NAS4 were significantly altered in the double mutant.NPF5.9 and NPF5.8 were shown to also mediate nitrate uptake and transport,although nitrate and Fe application did not reciprocally affect each other.Our findings uncovered the novel function of NPF5.9 and NPF5.8 in long-distance Fe transport and homeostasis,and further indicated that they possibly mediate nitrate transport and Fe homeostasis independently in Arabidopsis.

Targeting γ-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models

Background:We recently demonstrated an endolysosomal accumulation of theβ-secretase-derived APP C-terminal fragment(CTF)C99 in brains of Alzheimer disease(AD)mouse models.Moreover,we showed that the treatment with theγ-secretase inhibitor(D6)led to further increased endolysosomal APP-CTF levels,but also revealed extracellular APP-CTF-associated immunostaining.We here hypothesized that this latter staining could reflect extracellular vesicle(EV)-associated APP-CTFs and aimed to characterize theseγ-secretase inhibitor-induced APPCTFs.Methods:EVs were purified from cell media or mouse brains from vehicle-or D6-treated C99 or APPswedish expressing cells/mice and analyzed for APP-CTFs by immunoblot.Combined pharmacological,immunological and genetic approaches(presenilin invalidation and C99 dimerization mutants(GXXXG))were used to characterize vesicle-containing APP-CTFs.Subcellular APP-CTF localization was determined by immunocytochemistry.Results:Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains.Surprisingly,EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight(HMW)APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts.Accordingly,the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869.By combined pharmacological,immunological and genetic approaches,we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83.Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network,whereas oligomers were confined to endosomes and lysosomes,thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs.The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due toγ-secretase blockade,since it similarly occurred in presenilin-deficient fibroblasts.Further,our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis,γ-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN.Conclusions:This is the first study to demonstrate the presence of oligomeric APP-CTFs in AD mouse models,the levels of which are selectively enriched in endolysosomal compartments including exosomes and amplified byγ-secretase inhibition.Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset,progression and spreading.

Sulfuretin exerts diversified functions in the processing of amyloid precursor protein

Sulfuretin is a flavonoid that protects cell from damage induced by reactive oxygen species and inflammation.In this study,we investigated the role of sulfuretin in the processing of amyloid precursor protein(APP),in association with the two catalytic enzymes the a-secretase a disintegrin and metalloproteinase(ADAM10),and the beta-site APP cleaving enzyme 1(BACE1)that play important roles in the generation of β amyloid protein(Aβ)in Alzheimer’s disease(AD).We found that sulfuretin increased the levels of the immature but not the mature form of ADAM10 protein.The enhanced ADAM10 transcription by sulfuretin was mediated by the nucleotides444 to300 in the promoter region,and was attenuated by silencing or mutation of transcription factor retinoid X receptor(RXR)and by GW6471,a specific inhibitor of peroxisome proliferator-activated receptor α(PPAR-α).We further found that sulfuretin preferentially increased protein levels of the immature form of APP(im-APP)but significantly reduced those of BACE1,sAPPβ and β-CTF,whereas Ab1-42 levels were slightly increased.Finally,the effect of sulfuretin on BACE1 and im-APP was selectively attenuated by the translation inhibitor cycloheximide and by lysosomal inhibitor chloroquine,respectively.Taken together,(1)RXR/PPAR-α signaling was involved in sulfuretin-mediated ADAM10 transcription.(2)Alteration of Aβ protein level by sulfuretin was not consistent with that of ADAM10 and BACE1 protein levels,but was consistent with the elevated level of im-APP protein,suggesting that im-APP,an isoform mainly localized to trans-Golgi network,plays an important role in Ab generation.