Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies

Gireud‐Goss, Monica; Reyes-Esteves, Sahily; Wilson, M P; Farley, Madeline M.; Memarzadeh, Kimiya; Srinivasan, Saipraveen; Sirisaengtaksin, Natalie; Yamashita, Shinji; Tsunoda, Susan; Lang, Frederick F.; Waxham, M. Neal; Bean, Andrew J.

doi:10.1016/j.yexcr.2018.08.027

Cited by 6 publications

(18 citation statements)

References 94 publications

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“…The MVB sorting pathway plays a key role in protein degradation [26][27][28]. ESCRT, the key unit in the MVB sorting pathway, binds to endosomes to sort proteins for protein degradation or to transport to other organelles.…”

Section: Discussionmentioning

confidence: 99%

Enhanced β-carotene production by promoting the multivesicular body (MVB) pathway in Yarrowia lipolytica

Yang¹,

Liu²,

Shan³

et al. 2020

Preprint

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Background: β-carotene is a precursor of vitamin A and has great commercial value as an additive in foods and feeds. Many pathways not directly related to the β-carotene synthesis affect β-carotene production since the interactions among metabolic fluxes of cells confer a complex regulatory network. Engineered Y. lipolytica strain has excellent potential for β-carotene production as oleaginous yeast. Optimizing indirectly metabolic pathways in Y. lipolytica may offer a new strategy for making the β-carotene production achieve a commercially viable yield. Results:In this study, we found that the proper promotion of the multivesicular body (MVB) sorting pathway elevated the production of β-carotene by 1.58 fold when overexpressing one copy of the Did2 gene in Y. lipolytica. Through the measurement of ATP, NADPH, the mRNA, and protein level of key genes in the β-carotene synthesis pathway, the reason for β-carotene elevated was deuced that the protein level of the key enzymes (tHMG and CarA) was increased. When overexpressing two copies of the Did2 gene, the transcription level of the key genes was all elevated. However, the protein level of key enzymes in the β-carotene synthesis pathway was reduced compared with the overexpressing one copy of the Did2 gene, which resulted in reduced β-carotene content. Conclusion:This study suggests that the MVB sorting pathway is not responsible for sorting protein but has a crucial regulating effect on protein abundance in cells. Engineering the MVB sorting pathway could potentially increase the production of other high-value products. Moreover, manipulation of indirectly related metabolic pathways also is a critical strategy in synthetic biology research.

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Section: Discussionmentioning

confidence: 99%

Enhanced β-carotene production by promoting the multivesicular body (MVB) pathway in Yarrowia lipolytica

Yang¹,

Liu²,

Shan³

et al. 2020

Preprint

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“…Mammalian and S. cerevisiae cytosol as well as cell-free reconstitution assays were performed as previously described in Gireud-Goss et al 53 For cell-free reconstitution experiments testing dependence of EGFR and EGFRvIII on cholesterol, following the nal centrifugation step (90,000 x g for 30 minutes) endosomes used in reactions including Methyl-β cyclodextrin (MβCD) (Sigma-Aldrich) were incubated with 20mM MβCD at 37°C for 15 minutes. The endosomes were then centrifuged at 90,000 x g for 30 minutes, and resulting endosomes were incorporated into standard reactions.…”

Section: Cytosol Preparation and Cell-free Reconstitutionmentioning

confidence: 99%

“…Reactions either contained U87 cytosol, U87 cytosol with 20mM MβCD, or U87 cytosol with 20mM MβCD and 1mM cholesterol (Sigma-Aldrich). All reactions were adjusted to have a nal volume of 50 μL as described in Gireud-Goss et al 53 Criteria for Calculating Reaction E ciency in Cell-Free Sorting Assay All experimental reactions were normalized to starting endosomal controls to obtain the reaction e ciency. To obtain reaction e ciency, the amount of EGFR on starting membranes added to each reaction was compared to the amount of EGFR in reactions that contained membranes, cytosol, ATP and had been cleaved by trypsin.…”

Section: Cytosol Preparation and Cell-free Reconstitutionmentioning

confidence: 99%

“…The epitope is then detected via immunoblotting following reaction completion. If the epitope remains exposed to the extracellular environment, as would occur if the membrane protein was not internalized from the endosomal membrane into the ILV, it would be digested by exogenous trypsin and would no longer be detectable, indicating that the receptor remained on the limiting membrane of the endosome [52][53][54] .…”

Section: Egfrviii Is Internalized Into Intraluminal Vesicles Of the Mmentioning

confidence: 99%

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Endosomal trafficking of the EGFR mutant, EGFRvIII, results in exosomal secretion that triggers astrocyte reactivity

Memarzadeh

Patrizz²,

Koellhoffer³

et al. 2019

Preprint

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Background: The epidermal growth factor receptor (EGFR) variant three (EGFRvIII) mutation is linked with approximately one third of Glioblastoma Multiforme (GBM) tumors and is associated with poor patient prognosis. Persistent signaling due to a lack of the EGFR ectodomain and inefficient degradation have been suggested to underlie the tumorgenic properties of EGFRvIII. Methods: Cell viability and trans-well migration assays were used to determine the effects that expression of the oncoprotein, EGFRvIII, had on glioma cells. A cell-free reconstitution assay developed by our laboratory was utilized to determine trafficking of EGFR and EGFRvIII at the late endosome and determine molecular requirements for inward budding of proteins into the MVB. Western Blot Analysis and Nanosight Tracking Analysis were used to characterize exosomes by protein marker presence and vesicle size, respectively. Immunohistochemistry and Western Blot analysis were used to determine astrocyte reactivity marked by GFAP expression. Results: Like the parental EGFR, we observed that EGFRvIII is internalized into the intraluminal vesicles of late endosomes / multivesicular bodies (MVBs) but does not follow the canonical pathway by which wild-type EGFR is degraded following MVB fusion with lysosomes. These studies suggested that EGFRvIII is secreted on exosomes, the intraluminal vesicles that are secreted upon MVB fusion with the plasma membrane, suggested that EGFRvIII is localized in a subset of MVBs that preferentially fuse with the plasma membrane rather than with lysosomes which may account for its decreased degradation. Astrocytes are a component of the GBM tumor microenvironment with which tumor cells interact in a paracrine manner. EGFRvIII-containing exosomes derived from GBM cells induce changes in astrocytes that mimic reactive astrogliosis including an increase in glial fibrillary acidic protein (GFAP). Conclusions: These studies reveal novel aspects of the endocytic trafficking of EGFRvIII that underlie its reduced degradation and the mechanism by which it is packaged into exosomes for secretion. Moreover, EGFRvIII secretion on exosomes can facilitate changes in the tumor microenvironment to enable tumor growth.

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“…[16][17][18][19] Ubiquitination of membrane proteins on the limiting endosomal membrane regulates internalization into internal vesicles (ILVs) of the MVB, 12,20 an important step that determines the subsequent itinerary of the EGFR. [21][22][23][24][25] EGFR ubiquitination and subsequent lysosomal degradation can regulate the amount of EGFR on the cell surface and thereby modulates EGFR-mediated signaling. 7 Interestingly, EGFR inhibitors have not proven efficacious in neuroblastoma patients.…”

Section: Introductionmentioning

confidence: 99%

Low UBE4B expression increases sensitivity of chemoresistant neuroblastoma cells to EGFR and STAT5 inhibition

Memarzadeh

Savage

Bean

2019

Cancer Biology & Therapy

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Neuroblastoma is the most common malignancy in infants. Overexpression of the epidermal growth factor receptor (EGFR) in neuroblastoma tumors underlies resistance to chemotherapeutics. UBE4B, an E3/E4 ubiquitin ligase involved in EGFR degradation, is located on chromosome 1p36, a region in which loss of heterozygosity is observed in approximately one-third of neuroblastoma tumors and is correlated with poor prognosis. In chemoresistant neuroblastoma cells, depletion of UBE4B yielded significantly reduced cell proliferation and migration, and enhanced apoptosis in response to EGFR inhibitor, Cetuximab. We have previously shown that UBE4B levels are inversely correlated with EGFR levels in neuroblastoma tumors. We searched for additional targets of UBE4B that mediate cellular alterations associated with tumorogenesis in chemoresistant neuroblastoma cells depleted of UBE4B using reverse phase protein arrays. The expression of STAT5a, an effector protein downstream of EGFR, doubled in the absence of UBE4B, and verified by quantitative immunoblotting. Chemoresistant neuroblastoma cells were treated with SH-4-54, a STAT5 inhibitor, and observed insignificant effects on cell proliferation, migration, and apoptosis. However, SH-4-54 significantly enhanced the anti-proliferative and antimigratory effects of Cetuximab in naïve SK-N-AS neuroblastoma cells. Interestingly, in UBE4B depleted SK-N-AS cells, SH-4-54 significantly potentiated the effect of Cetuximab rendering cells increasingly sensitive an otherwise minimally effective Cetuximab concentration. Thus, neuroblastoma cells with low UBE4B levels were significantly more sensitive to combined EGFR and STAT5 inhibition than parental cells. These findings may have potential therapeutic implications for patients with 1p36 chromosome LOH and low tumor UBE4B expression.

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Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies

Cited by 6 publications

References 94 publications

Enhanced β-carotene production by promoting the multivesicular body (MVB) pathway in Yarrowia lipolytica

Enhanced β-carotene production by promoting the multivesicular body (MVB) pathway in Yarrowia lipolytica

Endosomal trafficking of the EGFR mutant, EGFRvIII, results in exosomal secretion that triggers astrocyte reactivity

Low UBE4B expression increases sensitivity of chemoresistant neuroblastoma cells to EGFR and STAT5 inhibition

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