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1.
Virol Sin ; 37(3): 321-330, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1813214

ABSTRACT

Retromer and sorting nexins (SNXs) transport cargoes from endosomes to the trans-Golgi network or plasma membrane. Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses, including members of Coronaviridae, Flaviviridae and Retroviridae. Key components of retromer/SNXs, such as Vps35, Vps26, SNX5 and SNX27, can affect multiple steps of the viral life cycle, including facilitating the entry of viruses into cells, participating in viral replication, and promoting the assembly of virions. Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus, which will shed mechanistic insights into controlling virus infection.


Subject(s)
Sorting Nexins , Viruses , Animals , Life Cycle Stages , Protein Transport , Sorting Nexins/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism
2.
Oxid Med Cell Longev ; 2022: 9366494, 2022.
Article in English | MEDLINE | ID: covidwho-1807713

ABSTRACT

Trehalose, a natural disaccharide, is synthesized by many organisms when cells are exposed to stressful stimuli. On the basis of its ability to modulate autophagy, trehalose has been considered an innovative drug for ameliorating many diseases, but its molecular mechanism is not well described. Previous findings demonstrated that trehalose plays a photoprotective role against ultraviolet (UV) B-induced damage through autophagy induction in keratinocytes. In this study, coimmunoprecipitation, label-free quantitative proteomic and parallel reaction monitoring, and western blot analysis demonstrated that trehalose promotes the interaction between tissue inhibitor of metalloproteinase (TIMP) 3 and Beclin1. Western blot and immunofluorescence staining analysis suggested that trehalose increases ATG9A localization in lysosomes and decreases its localization in the endoplasmic reticulum. Furthermore, in the presence or absence of UVB radiation, we evaluated the influence of TIMP3 and ATG9A small interfering RNA (siRNA) on the effect of trehalose on autophagy, cell death, migration, or interleukin-8 expression in keratinocytes, including HaCaT, A431, and human epidermal keratinocytes. The results revealed that in HaCaT cells, TIMP3 and ATG9A siRNA resulted in attenuation of trehalose-induced autophagy and inhibited cell death. In A431 cells, TIMP3 and ATG9A siRNA led to attenuation of trehalose-induced autophagy and cell death and inhibited migration. In human epidermal keratinocytes, trehalose-induced autophagy and inhibition of the interleukin-8 expression were blocked by ATG9A but not TIMP3 siRNA. In addition, the results of quantitative real-time PCR and immunohistochemistry analysis demonstrated the abnormal expression of TIMP3 and ATG9A in actinic keratosis and cutaneous squamous cell carcinoma skin tissues. These findings suggest the protective effects of trehalose in normal keratinocytes and its inhibitory effects on cancerous keratinocytes, possibly mediated by activation of autophagy and regulation of TIMP3 and ATG9A, providing the mechanistic basis for the potential use of trehalose in the prevention or treatment of UVB-induced skin diseases.


Subject(s)
Carcinoma, Squamous Cell , Skin Neoplasms , Autophagy , Autophagy-Related Proteins/metabolism , Carcinoma, Squamous Cell/pathology , Humans , Interleukin-8/metabolism , Keratinocytes/metabolism , Membrane Proteins/metabolism , Proteomics , RNA, Small Interfering/metabolism , Skin Neoplasms/metabolism , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/metabolism , Tissue Inhibitor of Metalloproteinase-3/pharmacology , Trehalose/pharmacology , Ultraviolet Rays/adverse effects , Vesicular Transport Proteins/metabolism
3.
FEBS Lett ; 595(17): 2248-2256, 2021 09.
Article in English | MEDLINE | ID: covidwho-1326724

ABSTRACT

The endoplasmic reticulum transmembrane protein vesicle-associated membrane protein-associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT-like motifs. In this study, we investigated the interactions of eight peptides containing FFAT-like motifs with the VAP-A MSP domain (VAP-AMSP ) by solution NMR. Six of eight peptides are specifically bound to VAP-A. Furthermore, we found that the RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT-like motif which specifically binds to VAP-AMSP as well as other FFAT-like motifs. Our results will contribute to the discovery of new VAP interactors.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Peptides/chemistry , SARS-CoV-2/enzymology , Vesicular Transport Proteins/chemistry , Amino Acid Motifs , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Nuclear Magnetic Resonance, Biomolecular , Peptides/metabolism , Protein Binding , SARS-CoV-2/metabolism , Vesicular Transport Proteins/metabolism
4.
Nat Cell Biol ; 23(8): 846-858, 2021 08.
Article in English | MEDLINE | ID: covidwho-1309445

ABSTRACT

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Autophagosomes/metabolism , Autophagy-Related Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Immunoprecipitation , Membrane Proteins/genetics , Microscopy, Confocal , Protein Transport/physiology , Vesicular Transport Proteins/genetics
5.
Cells ; 10(4)2021 03 28.
Article in English | MEDLINE | ID: covidwho-1154291

ABSTRACT

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons in the substantia nigra, intraneuronal deposition of misfolded proteins known as Lewy bodies, and chronic neuroinflammation. PD can arise from monogenic mutations, but in most cases, the etiology is unclear. Viral infection is gaining increasing attentions as a trigger of PD. In this study, we investigated whether the PD-causative 620 aspartate (D) to asparagine (N) mutation in the vacuolar protein sorting 35 ortholog (Vps35) precipitated herpes simplex virus (HSV) infection. We observed that ectopic expression of Vps35 significantly reduced the proliferation and release of HSV-1 virions; the D620N mutation rendered Vps35 a partial loss of such inhibitory effects. Tetherin is a host cell protein capable of restricting the spread of encapsulated viruses including HSV-1 and SARS-Cov-2, both of which are implicated in the development of parkinsonism. Compared with cells overexpressing wildtype Vps35, cells expressing mutant Vps35 with D620N had less Tetherin on cell surfaces. Real-time and static cell imaging revealed that Tetherin recycled through Vps35-positive endosomes. Expression of Vps35 with D620N reduced endosomal dynamics and frequency of motile Tetherin-containing vesicles, a sign of defective production of recycling carriers. Our study suggests that the D620N mutation in Vps35 hinders Tetherin trafficking to cell surfaces and facilitates virus spread.


Subject(s)
Bone Marrow Stromal Antigen 2/metabolism , Parkinson Disease/metabolism , Parkinson Disease/virology , Simplexvirus/metabolism , Vesicular Transport Proteins/metabolism , COVID-19/virology , Cell Line, Tumor , Endosomes/metabolism , Humans , Mutation , Parkinson Disease/genetics , Protein Transport/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Simplexvirus/pathogenicity , Transfection , Vesicular Transport Proteins/genetics , Virus Replication/genetics
6.
Dev Cell ; 56(4): 427-442.e5, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-978254

ABSTRACT

Autophagy acts as a cellular surveillance mechanism to combat invading pathogens. Viruses have evolved various strategies to block autophagy and even subvert it for their replication and release. Here, we demonstrated that ORF3a of the COVID-19 virus SARS-CoV-2 inhibits autophagy activity by blocking fusion of autophagosomes/amphisomes with lysosomes. The late endosome-localized ORF3a directly interacts with and sequestrates the homotypic fusion and protein sorting (HOPS) component VPS39, thereby preventing HOPS complex from interacting with the autophagosomal SNARE protein STX17. This blocks assembly of the STX17-SNAP29-VAMP8 SNARE complex, which mediates autophagosome/amphisome fusion with lysosomes. Expression of ORF3a also damages lysosomes and impairs their function. SARS-CoV-2 virus infection blocks autophagy, resulting in accumulation of autophagosomes/amphisomes, and causes late endosomal sequestration of VPS39. Surprisingly, ORF3a from the SARS virus SARS-CoV fails to interact with HOPS or block autophagy. Our study reveals a mechanism by which SARS-CoV-2 evades lysosomal destruction and provides insights for developing new strategies to treat COVID-19.


Subject(s)
Autophagosomes/metabolism , COVID-19/metabolism , Lysosomes/metabolism , SNARE Proteins/metabolism , Viroporin Proteins/metabolism , Autophagy , Autophagy-Related Proteins/metabolism , COVID-19/virology , HEK293 Cells , HeLa Cells , Humans , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Vesicular Transport Proteins/metabolism , Viroporin Proteins/genetics
7.
Am J Physiol Lung Cell Mol Physiol ; 320(1): L158-L163, 2021 01 01.
Article in English | MEDLINE | ID: covidwho-919085

ABSTRACT

Lungs of smokers and chronic obstructive pulmonary disease (COPD) are severely compromised and are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attack. The dangerous combination of enhanced SARS-CoV-2 attachment receptor protein ACE2 along with an increase in endocytic vacuoles will enable viral attachment, entry, and replication. The objective of the study was to identify the presence of SARS-CoV-2 host attachment receptor angiotensin-converting enzyme-2 (ACE2) along with endocytic vacuoles, early endosome antigen-1 (EEA1), late endosome marker RAB7, cathepsin-L, and lysosomal associated membrane protein-1 (LAMP-1) as lysosome markers in the airways of smokers and COPD patients. The study design was cross-sectional and involved lung resections from 39 patients in total, which included 19 patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I or GOLD stage II COPD, of which 9 were current smokers with COPD (COPD-CS) and 10 were ex-smokers with COPD (COPD-ES), 10 were normal lung function smokers, and 10 were never-smoking normal controls. Immunostaining for ACE2, EEA1, RAB7, and cathepsin-L was done. A comparative description for ACE2, EEA1, RAB7, and cathepsin-L expression pattern is provided for the patient groups. Furthermore, staining intensity for LAMP-1 lysosomes was measured as the ratio of the LAMP-1-stained areas per total area of epithelium or subepithelium, using Image ProPlus v7.0 software. LAMP-1 expression showed a positive correlation to patient smoking history while in COPD LAMP-1 negatively correlated to lung function. The active presence of ACE2 protein along with endocytic vacuoles such as early/late endosomes and lysosomes in the small airways of smokers and COPD patients provides evidence that these patient groups could be more susceptible to COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Pulmonary Disease, Chronic Obstructive/pathology , Smoking/pathology , Transport Vesicles/metabolism , Cathepsin L/metabolism , Cross-Sectional Studies , Disease Susceptibility , Humans , Lung/pathology , Lysosome-Associated Membrane Glycoproteins/metabolism , SARS-CoV-2 , Smokers , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism
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