Unconventional protein secretion-mediated protein quality control in health and diseases

健康和疾病中非常规蛋白质分泌介导的蛋白质质量控制

基本信息

批准号：
10697851
负责人：
Yihong Ye
金额：
$ 66.94万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10697851
关键词：
2019-nCoV 3-Dimensional Actins Affinity Alternative Therapies Astrocytes Binding Biological Assay Brain COVID-19 pandemic Cell Surface Receptors Cell membrane Cell surface Cells Charge Chemicals Classification Complement Complex DNA Topoisomerases Development Disease Embryo Endocytosis Endoplasmic Reticulum FDA approved Family Gene Expression Profile Gene Expression Profiling Glycosaminoglycans Goals Golgi Apparatus Health Heparan Sulfate Proteoglycan Heparitin Sulfate Human In Vitro Infection Inflammatory Integrins Label Lewy Bodies Ligation Lung Lysosomes MAPT gene Mammalian Cell Mediating Membrane Membrane Proteins Metalloproteases Methods Mitoxantrone Modeling Network-based Neurodegenerative Disorders Neuroglia Neurons Nucleosomes Parkinson Disease Pathologic Pathology Pathway interactions Patients Pattern Peptide Signal Sequences Pharmaceutical Preparations Physiological Process Protein Export Pathway Protein Secretion Proteins Proteomics Quality Control Reporting Research Project Grants SARS-CoV-2 entry inhibitor SARS-CoV-2 spike protein Signal Transduction Subgroup Surface System Tauopathies Tissues Topoisomerase Inhibitors Toxic effect Training Transplantation Up-Regulation Vaccines Viral Virus Virus Diseases Work alpha synuclein analog base breakthrough infection chemokine cytokine cytotoxicity dopaminergic neuron dorsal motor nucleus drug repurposing extracellular graph neural network inhibitor knock-down late endosome misfolded protein neuron loss neurotoxic new therapeutic target olfactory bulb polypeptide pre-formed fibril prion hypothesis protein aggregation proteotoxicity receptor recruit response sensor synuclein tau Proteins tau aggregation transcriptome sequencing transmission process uptake viral entry inhibitor virtual

项目摘要

Proteinaceous inclusions termed Lewy bodies (LBs) are a classical hallmark of Parkinson's disease (PD). The primary component of these inclusions is alpha-synuclein (a-syn), a protein with an intrinsic propensity to misfold and aggregate. In PD patients, alpha-syn inclusions first observed in the olfactory bulb and the dorsal motor nucleus, progressively spread throughout the brain. Further findings that healthy embryonic dopamine neurons transplanted into PD patients developed LBs points suggest the tantalizing possibility of neuron-to-neuron transmission of a-syn. Subsequent work confirmed that synthetic a-syn pre-formed fibrils (PFFs) can be taken up by neurons, eliciting the misfolding of endogenous -syn into insoluble Lewy-like inclusions. Collectively, these studies led to the prion hypothesis of PD, wherein misfolded -syn provides a template for seeding new aggregates, propagating misfolded a-syn, and associated cytotoxicity. Thus, the propagation of alpha-syn is a viable new target to be explored in the development of new PD therapies. The intercellular transmission of synuclein consists of two key steps: the secretion of synuclein from a donor neuron and its uptake by a recipient neuron. Misfolding-associated protein secretion (MAPS) is a recently discovered protein quality control process that selectively exports misfolded cytosolic proteins including a-syn. Secretion through MAPS requires the membrane-localized deubiquitinase USP19, which recruits aberrant polypeptides to the endoplasmic reticulum (ER) surface to facilitate their incorporation into late endosomes that are in tight association with the ER. Misfolded proteins are secreted to the extracellular milieu when late endosomes fuse with the plasma membrane. The fate of the released misfolded proteins is currently unknown. Our recent studies suggest that mammalian cells can internalize misfolded proteins via endocytosis, but it is unclear whether they possess one or more receptors for misfolded proteins. Whether internalized proteins can impose damage prior to degradation by the lysosome is also unclear. The proposal is to elucidate the physiological relevance of the MAPS pathway by characterizing the interplay between secreted misfolded proteins and target cells. Using a proximity-based ligation approach, we have used purified Tau as a bait to identify candidate Protein aggregates formed by microtubule-associated protein Tau can be transmitted in a stereotypic pattern in human brains, which correlates with the progression of several neurodegenerative diseases collectively termed Tauopathies. This process requires Tau, released from neurons, to interact with a cell surface receptor on a target cell, but little is known about the underlying mechanisms and the downstream pathophysiological consequences, particularly for Tau that engages glial cells. Using a spatially resolved proteomic mapping strategy, we identify integrin V/1, a heterodimer of the integrin family, as a receptor that not only mediates Tau fibril entry into astrocytes but also activates integrin signaling upon Tau binding. We show that distinct Tau species differentially activate integrin signaling, with filamentous Tau being a more robust stimulator. This leads to NFB activation and differential upregulation of pro-inflammatory cytokines and chemokines. Additionally, a sub-group of neurotoxic astrocyte markers are also induced in an integrin-dependent manner preferentially by filamentous Tau, causing astrocytes to release a neurotoxic factor(s). Together, these findings establish a paradigm that astrocytes can be directly converted into a neurotoxic state by filamentous Tau via an integrin receptor, which as a sensor transducing unfolded protein pathology, may provide a new therapeutic target for Tauopathies. In a more recent effort, we used RNAseq to analyze the gene expression profile changes in response to a proteotoxic insult using tau fibrils as a model. We find that in addition to integrin signing, tau fibril ligation to an integrin receptor also activates PI3K, which leads to a cellular response that is distinct from the canonical integrin signaling. We further analyzed the secretome of cells treated with tau fibrils and found that many proteins are secreted in a tau-inducible manner. Among the secreted proteins, we identified C3 complement and Mmp3 metalloprotease as key regulators of neuronal cell death. This study reveals why astrocytes stimulated by tau fibrils can generate a pathological response via a physiologically critical cell surface receptor. We recently reported that the infection of human cells by SARS-CoV-2, the virus underlying the ongoing COVID-19 pandemic depends on an endocytosis mechanism akin to the entry of alpha-Syn fibrils. Accordingly, knockdown of cell surface HSPG or cortex actin reduces viral infection in an in vitro cell-based model. A drug repurpose screen has identified several FDA-approved drugs that can effectively block the entry of SARS-CoV-2 in vitro. We further showed that in addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of Spike with a downstream receptor to promote viral entry. We reported that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a heparan sulfate-spike complex to compromise the fusogenic function of Spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar heparan sulfate-binding activities but with reduced affinity for DNA topoisomerases may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era. To identify additional viral entry inhibitors, we also developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in 2000 potential hits. A hit confirmation assay with the uptake of a fluorescently labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to the strong inhibitory activity

称为路易体 (LB) 的蛋白质内含物是帕金森病 (PD) 的经典标志。这些内含物的主要成分是 α-突触核蛋白 (a-syn)，这是一种具有错误折叠和聚集内在倾向的蛋白质。在帕金森病患者中，α-syn 内含物首先在嗅球和背运动核中观察到，随后逐渐扩散到整个大脑。进一步的发现表明，移植到帕金森病患者体内的健康胚胎多巴胺神经元产生了 LB 点，这表明神经元间 a-syn 传递的可能性是诱人的。随后的工作证实，合成的α-syn预形成原纤维（PFF）可以被神经元吸收，引起内源性α-syn错误折叠成不溶性的路易样内含物。总的来说，这些研究提出了PD的朊病毒假说，其中错误折叠的-syn提供了播种新聚集体、传播错误折叠的a-syn和相关细胞毒性的模板。因此，α-syn 的传播是新 PD 疗法开发中需要探索的一个可行的新靶点。突触核蛋白的细胞间传递由两个关键步骤组成：供体神经元分泌突触核蛋白以及受体神经元摄取突触核蛋白。错误折叠相关蛋白分泌 (MAPS) 是最近发现的一种蛋白质质量控制过程，可选择性输出包括 a-syn 在内的错误折叠胞浆蛋白。通过 MAPS 的分泌需要膜定位的去泛素酶 USP19，它将异常的多肽招募到内质网 (ER) 表面，以促进它们掺入与 ER 紧密相关的晚期内涵体中。当晚期内体与质膜融合时，错误折叠的蛋白质被分泌到细胞外环境。释放的错误折叠蛋白质的命运目前未知。我们最近的研究表明，哺乳动物细胞可以通过内吞作用内化错误折叠的蛋白质，但尚不清楚它们是否拥有一种或多种错误折叠蛋白质的受体。内化蛋白是否会在被溶酶体降解之前造成损害还不清楚。该提案旨在通过表征分泌的错误折叠蛋白与靶细胞之间的相互作用来阐明 MAPS 途径的生理相关性。通过基于邻近的连接方法，我们使用纯化的 Tau 作为诱饵来识别由微管相关蛋白 Tau 形成的候选蛋白聚集体，可以在人脑中以刻板模式传播，这与几种统称为神经退行性疾病的进展相关。陶氏病。这一过程需要从神经元释放的 Tau 蛋白与靶细胞上的细胞表面受体相互作用，但人们对其潜在机制和下游病理生理学后果知之甚少，特别是对于与神经胶质细胞结合的 Tau 蛋白。使用空间分辨蛋白质组图谱策略，我们将整合素 V/1（整合素家族的一种异二聚体）鉴定为受体，不仅介导 Tau 原纤维进入星形胶质细胞，而且在 Tau 结合时激活整合素信号传导。我们发现不同的 Tau 物种会差异性地激活整合素信号传导，其中丝状 Tau 是更强大的刺激剂。这导致 NFB 激活以及促炎细胞因子和趋化因子的差异上调。此外，神经毒性星形胶质细胞标记物的亚组也优先由丝状 Tau 以整合素依赖性方式诱导，导致星形胶质细胞释放神经毒性因子。总之，这些发现建立了一个范例，即星形胶质细胞可以通过整合素受体被丝状 Tau 直接转化为神经毒性状态，作为转导未折叠蛋白病理学的传感器，可能为 Tau 病提供新的治疗靶点。在最近的一项工作中，我们使用 RNAseq 以 tau 原纤维为模型来分析响应蛋白毒性损伤的基因表达谱变化。我们发现，除了整合素信号之外，tau 原纤维与整合素受体的连接也会激活 PI3K，从而导致与典型整合素信号传导不同的细胞反应。我们进一步分析了用 tau 原纤维处理的细胞的分泌组，发现许多蛋白质以 tau 诱导的方式分泌。在分泌的蛋白质中，我们发现 C3 补体和 Mmp3 金属蛋白酶是神经元细胞死亡的关键调节因子。这项研究揭示了为什么 tau 原纤维刺激的星形胶质细胞可以通过生理上关键的细胞表面受体产生病理反应。我们最近报道说，SARS-CoV-2（导致持续的 COVID-19 大流行的病毒）对人类细胞的感染依赖于类似于 α-Syn 原纤维进入的内吞机制。因此，在体外细胞模型中，细胞表面 HSPG 或皮质肌动蛋白的敲低可减少病毒感染。药物再利用筛选已确定了几种 FDA 批准的药物，可以在体外有效阻止 SARS-CoV-2 的进入。我们进一步表明，除了蛋白质受体外，SARS-CoV-2刺突（S）蛋白还与硫酸乙酰肝素相互作用，硫酸乙酰肝素是一种带负电荷的糖胺聚糖（GAG），附着在细胞表面的某些膜蛋白上。这种相互作用促进了 Spike 与下游受体的结合，从而促进病毒进入。我们报道了米托蒽醌（Mitoxantrone）是 FDA 批准的拓扑异构酶抑制剂，其靶向硫酸乙酰肝素-刺突复合物，以损害刺突在病毒进入中的融合功能。作为单一药物，米托蒽醌可抑制细胞模型和人肺 EpiAirway 3D 组织中真正的 SARS-CoV-2 毒株的感染。基因表达谱支持质膜作为米托蒽醌的主要靶标，但也强调了针对核小体动力学的不良活性。我们提出，米托蒽醌类似物具有类似的硫酸乙酰肝素结合活性，但对 DNA 拓扑异构酶的亲和力降低，可能会提供一种替代疗法来克服后疫苗时代的突破性感染。为了识别其他病毒进入抑制剂，我们还使用从实验识别的 HSPG 和肌动蛋白抑制剂中提取的信息开发并训练了基于图卷积网络 (GCN) 的分类模型。这种方法使我们能够虚拟筛选 170,000 种化合物，从而产生 2000 种潜在的命中结果。通过摄取荧光标记的 HSPG 货物进行命中确认测定，进一步筛选出 256 种活性化合物。其中，16个化合物具有中等到强的抑制活性