GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Ahat, Erpan; Bui, Sarah; Zhang, J.; Leprevost, Felipe da Veiga; Sharkey, Lisa M.; Reid, Whitney; Nesvizhskii, Alexey I.; Paulson, Henry L.; Wang, Yanzhuang

doi:10.1016/j.jbc.2022.102219

Cited by 21 publications

(28 citation statements)

References 65 publications

(102 reference statements)

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“…These findings highlight the functional role of GRASP55 in unconventional protein secretion as the positive regulator of secretory autophagosome formation. This is different from a previous report in which GRASP55 was found to mediate autophagy-dependent secretion of mutant huntingtin by promoting autophagosome-lysosome fusion (autophagic flux) 71 .…”

Section: Discussioncontrasting

confidence: 99%

“…Although GRASP55 was initially characterized as Golgi reassembly and stacking protein, later studies indicated that it is also involved in unconventional trafficking of cytosolic and transmembrane proteins 32,71,[84][85][86][87] . Notably, evidence supporting GRASP55's involvement in unconventional secretion of inflammatory mediators is mounting 34,40,42,88 while the mechanisms remain to be elucidated.…”

Section: Discussionmentioning

confidence: 99%

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Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

Bhushan,

Chan,

Sun

et al. 2024

Preprint

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Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatoryP. aeruginosaculture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1α, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II by confocal microscopy. Mechanistically, mass spectrometric analysis of K6a immunoprecipitates identified Sec16A, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

Bhushan,

Chan,

Sun

et al. 2024

Preprint

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“…As expected, GRASP55 preferentially pulled down Golgin-45, whereas GRASP65 significantly enriched GM130. As in previous reports, we also found several members of the p24/transmembrane emp24 domain family of cargo receptors (TMED) in a complex with GRASP55 (Barr et al, 2001; Ahat et al, 2022). Strikingly, mass spectrometry analysis further identified several RNA-binding proteins (RBPs) that specifically associate with GRASP65, including G3BP1 (G3BP Stress Granule Assembly Factor 1), FXR1 (Fragile X mental retardation syndrome-related protein 1), CAPRIN1 (also known as RNA granule protein 105 or RNG105), and PABPC1 (PABP/Polyadenylate-binding protein 1).…”

Section: Resultssupporting

confidence: 88%

RNA scaffolds the Golgi ribbon by forming condensates with GM130

Zhang

Seemann

2023

Preprint

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The mammalian Golgi apparatus is composed of stacks of cisternae that are laterally linked to form a continuous ribbon like structure, but the molecular mechanisms that maintain the Golgi ribbon remain unclear. Here, we show that ribbon formation is mediated by biomolecular condensates of RNA and the Golgi resident protein GM130. We identified GM130 as a membrane-bound RNA binding protein at the Golgi. Acute degradation of either RNA or GM130 in cells disrupted the Golgi ribbon. Under stress conditions, RNA was displaced from GM130 and the ribbon was disjoint, which was restored after cells recovered from stress. When overexpressed in cells, GM130 formed RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its N-terminus, which was sufficient to recruit RNA to drive condensate assemblyin vitro. Condensates of the N-terminal domain of GM130 and RNA were sufficient to link purified rat liver Golgi membranes which is a reconstruction of aspects of lateral linking of stacks into a ribbon-like structure. Together, these studies reveal that biomolecular condensates of GM130-RNA scaffold the Golgi ribbon.

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“…Cell lysates were resolved on precast 12% Bis-Tris NuPAGE gels (Invitrogen), transferred to PVDF membranes (Bio-Rad), and blotted with primary antibodies (see below and Supplemental Table 2 ) followed by species-specific HRP-conjugated secondary antibody. Primary antibodies (see Supplemental Table 2 ) include surfactant protein B (PT3, a rabbit polyclonal antibody against bovine SFTPB (1:2,500) ( 74 ), mature SFTPC (1:1,000, Seven Hills Bioreagents), and hexokinase 1 (1:2,500, Proteintech) used as the loading control ( 75 ). Visualization was accomplished using the Odyssey Imaging System (LICOR Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Culture impact on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells

Alysandratos¹,

Alba²,

Yao³

et al. 2023

JCI Insight

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Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we perform head-to-head comparisons between the transcriptomes of fresh primary (1 o ) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). We find each population occupies a distinct transcriptomic space with cultured AEC2s (1 o and iAEC2s) exhibiting similarities to and differences from freshly purified 1 o cells. Across each cell type, we find an inverse relationship between proliferative and maturation states, with pre-culture 1 o AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2 do not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s co-cultured with fibroblasts acquires a "transitional cell state" described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1 o and engineered AEC2s, two in vitro models that can be harnessed to study human lung health and disease.

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GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Cited by 21 publications

References 65 publications

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

RNA scaffolds the Golgi ribbon by forming condensates with GM130

Culture impact on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells

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