ABSTRACT
Over the past decade, immunotherapies have brought about significant changes in how we approach the treatment of various solid tumors and blood-related cancers. However, the effectiveness of checkpoint blockade therapy has been constrained to a rate of under 30â¯%. A significant challenge in the realm of tumor immunotherapy revolves around comprehending the mechanisms through which regulatory T (Treg) cells induce immunosuppression. We have recently discovered that USP22 (ubiquitin-specific peptidase 22) a deubiquitinating enzyme that is increased in various tumors, is an oncogene and controls Treg immune suppressive activity for tumor evasion, providing a rationale for USP22 targeting to achieve both onco- and immuno-therapeutic efficacies. Herein, we identified the traditional Chinese secoiridoid compound gentiopicroside as a USP22 inhibitor. Gentiopicroside treatment decreased the forkhead box P3 (Foxp3) expression, which subsequently reduced Treg immune suppressive activity. Treatment of cancer cells by gentiopicroside resulted in an increase in histone 2B monoubiquitination (H2Bub) in a USP22-dependent manner and a decrease in programmed cell death ligand 1 (PD-L1) expression, both of which are known as USP22-specific substrates. Docking and molecular dynamic simulation revealed that gentiopicroside stably binds to USP22 catalytic pocket, supporting that gentiopicroside is a USP22 inhibitor. Importantly, administration of gentiopicroside to mice significantly inhibited the growth of syngenetic lung adenocarcinoma. Further analysis of intratumoral immune cells revealed a dramatic increase CD8+ T cell production of IFN-γ and granzyme B (GZMB), confirming that gentiopicroside enhances antitumor immunity. Our study revealed that gentiopicroside is a USP22-specific inhibitor with potent antitumor therapeutic potentials.
Subject(s)
Immunotherapy , Iridoid Glucosides , T-Lymphocytes, Regulatory , Ubiquitin Thiolesterase , Iridoid Glucosides/pharmacology , Humans , Animals , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Immunotherapy/methods , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/antagonists & inhibitors , Mice , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Mice, Inbred BALB C , Molecular Docking Simulation , B7-H1 Antigen/metabolism , B7-H1 Antigen/antagonists & inhibitors , Forkhead Transcription FactorsABSTRACT
Introduction: The rapid development of artificial intelligence (AI) in healthcare has exposed the unmet need for growing a multidisciplinary workforce that can collaborate effectively in the learning health systems. Maximizing the synergy among multiple teams is critical for Collaborative AI in Healthcare. Methods: We have developed a series of data, tools, and educational resources for cultivating the next generation of multidisciplinary workforce for Collaborative AI in Healthcare. We built bulk-natural language processing pipelines to extract structured information from clinical notes and stored them in common data models. We developed multimodal AI/machine learning (ML) tools and tutorials to enrich the toolbox of the multidisciplinary workforce to analyze multimodal healthcare data. We have created a fertile ground to cross-pollinate clinicians and AI scientists and train the next generation of AI health workforce to collaborate effectively. Results: Our work has democratized access to unstructured health information, AI/ML tools and resources for healthcare, and collaborative education resources. From 2017 to 2022, this has enabled studies in multiple clinical specialties resulting in 68 peer-reviewed publications. In 2022, our cross-discipline efforts converged and institutionalized into the Center for Collaborative AI in Healthcare. Conclusions: Our Collaborative AI in Healthcare initiatives has created valuable educational and practical resources. They have enabled more clinicians, scientists, and hospital administrators to successfully apply AI methods in their daily research and practice, develop closer collaborations, and advanced the institution-level learning health system.
ABSTRACT
While deubiquitinase ATXN3 has been implicated as a potential oncogene in various types of human cancers, its role in colon adenocarcinoma remains understudied. Surprisingly, our findings demonstrate that ATXN3 exerts an antitumor effect in human colon cancers through potentiating Galectin-9-induced apoptosis. CRISPR-mediated ATXN3 deletion unexpectedly intensified colon cancer growth both in vitro and in xenograft colon cancers. At the molecular level, we identified ATXN3 as a bona fide deubiquitinase specifically targeting Galectin-9, as ATXN3 interacted with and inhibited Galectin-9 ubiquitination. Consequently, targeted ATXN3 ablation resulted in reduced Galectin-9 protein expression, thereby diminishing Galectin-9-induced colon cancer apoptosis and cell growth arrest. The ectopic expression of Galectin-9 fully reversed the growth of ATXN3-null colon cancer in mice. Furthermore, immunohistochemistry staining revealed a significant reduction in both ATXN3 and Galectin-9 protein expression, along with a positive correlation between them in human colon cancer. Our study identifies the first Galectin-9-specific deubiquitinase and unveils a tumor-suppressive role of ATXN3 in human colon cancer.
Subject(s)
Adenocarcinoma , Apoptosis , Ataxin-3 , Colonic Neoplasms , Galectins , Humans , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Colonic Neoplasms/genetics , Galectins/metabolism , Galectins/genetics , Animals , Ataxin-3/metabolism , Ataxin-3/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/genetics , Mice , Cell Line, Tumor , Ubiquitination , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Repressor ProteinsABSTRACT
Antitumor responses of CD8+ T cells are tightly regulated by distinct metabolic fitness. High levels of glutathione (GSH) are observed in the majority of tumors, contributing to cancer progression and treatment resistance in part by preventing glutathione peroxidase 4-dependent (GPX4-dependent) ferroptosis. Here, we show the necessity of adenosine A2A receptor (A2AR) signaling and the GSH/GPX4 axis in orchestrating metabolic fitness and survival of functionally competent CD8+ T cells. Activated CD8+ T cells treated ex vivo with simultaneous inhibition of A2AR and lipid peroxidation acquire a superior capacity to proliferate and persist in vivo, demonstrating a translatable means to prevent ferroptosis in adoptive cell therapy. Additionally, we identify a particular cluster of intratumoral CD8+ T cells expressing a putative gene signature of GSH metabolism (GMGS) in association with clinical response and survival across several human cancers. Our study addresses a key role of GSH/GPX4 and adenosinergic pathways in fine-tuning the metabolic fitness of antitumor CD8+ T cells.
Subject(s)
Neoplasms , Receptor, Adenosine A2A , Humans , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Receptor, Adenosine A2A/genetics , Receptor, Adenosine A2A/metabolism , Neoplasms/genetics , Neoplasms/therapy , Neoplasms/metabolism , CD8-Positive T-Lymphocytes/metabolism , Glutathione/metabolismABSTRACT
Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. These viruses infect human host cells via the viral spike protein binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). Using over 1300 IgG sequences derived from convalescent patient B cells that bind with spike's receptor binding domain (RBD), we first established protein structural docking models in assessing the RBD-IgG-ACE2 interaction interfaces and predicting the virus-neutralizing activity of each IgG with a confidence score. Additionally, employing Gaussian process regression (also known as Kriging) in a latent space of an antibody language model, we predicted the landscape of IgGs' activity profiles against individual coronaviral variants of concern. With functional analyses and experimental validations, we efficiently prioritized IgG candidates for neutralizing a broad spectrum of viral variants (wildtype, Delta, and Omicron) to prevent the infection of host cells in vitro and hACE2 transgenic mice in vivo. Furthermore, the computational analyses enabled rational redesigns of selective IgG clones with single amino acid substitutions at the RBD-binding interface to improve the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screening and re-design even in low-data regimes combining protein language models and Kriging for antibody sequence analysis, activity prediction, and efficacy improvement, in synergy with physics-driven protein docking models for antibody-antigen interface structure analyses and functional optimization.
ABSTRACT
While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.
Subject(s)
Neoplasms , T-Lymphocytes, Regulatory , Humans , Animals , Mice , Mediator Complex Subunit 1/metabolism , Forkhead Transcription Factors , Neoplasms/pathology , Inflammation/metabolism , Tumor MicroenvironmentABSTRACT
Progress in sequencing technologies and clinical experiments has revolutionized immunotherapy on solid and hematologic malignancies. However, the benefits of immunotherapy are limited to specific patient subsets, posing challenges for broader application. To improve its effectiveness, identifying biomarkers that can predict patient response is crucial. Machine learning (ML) play a pivotal role in harnessing multi-omic cancer datasets and unlocking new insights into immunotherapy. This review provides an overview of cutting-edge ML models applied in omics data for immunotherapy analysis, including immunotherapy response prediction and immunotherapy-relevant tumor microenvironment identification. We elucidate how ML leverages diverse data types to identify significant biomarkers, enhance our understanding of immunotherapy mechanisms, and optimize decision-making process. Additionally, we discuss current limitations and challenges of ML in this rapidly evolving field. Finally, we outline future directions aimed at overcoming these barriers and improving the efficiency of ML in immunotherapy research.
ABSTRACT
As part of a symposium, current and former directors of Immune Monitoring cores and investigative oncologists presented insights into the past, present and future of immune assessment. Dr. Gnjatic presented a classification of immune monitoring technologies ranging from universally applicable to experimental protocols, while emphasizing the need for assay harmonization. Dr. Obeng discussed physiologic differences among CD8 T cells that align with anti-tumor responses. Dr. Lyerly presented the Soldano Ferrone lecture, commemorating the passionate tumor immunologist who inspired many, and covered a timeline of monitoring technology development and its importance to immuno-oncology. Dr. Sonabend presented recent achievements in glioblastoma treatment, accentuating the range of monitoring techniques that allowed him to refine patient selection for clinical trials. Dr. Guevara-Patiño focused on hypoxia within the tumor environment and stressed that T cell viability is not to be confused with functionality. Dr. Butterfield accentuated monitoring of dendritic cell metabolic (dys)function as a determinant for tumor vaccine success. Lectures were interspersed with select abstract presentations. To summarize the concepts, Dr. Maecker from Stanford led an informative forum discussion, pointing towards the future of immune monitoring. Immune monitoring continues to be a guiding light towards effective immunotherapeutic strategies.
ABSTRACT
Regulation of tumoral PD-L1 expression is critical to advancing our understanding of tumor immune evasion and the improvement of existing antitumor immunotherapies. Herein, we describe a CRISPR-based screening platform and identified ATXN3 as a positive regulator for PD-L1 transcription. TCGA database analysis revealed a positive correlation between ATXN3 and CD274 in more than 80% of human cancers. ATXN3-induced Pd-l1 transcription was promoted by tumor microenvironmental factors, including the inflammatory cytokine IFN-γ and hypoxia, through protection of their downstream transcription factors IRF1, STAT3, and HIF-2α. Moreover, ATXN3 functioned as a deubiquitinase of the AP-1 transcription factor JunB, indicating that ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells largely abolished IFN-γ- and hypoxia-induced PD-L1 expression and consequently enhanced antitumor immunity in mice, and these effects were partially reversed by PD-L1 reconstitution. Furthermore, tumoral ATXN3 suppression improved the preclinical efficacy of checkpoint blockade antitumor immunotherapy. Importantly, ATXN3 expression was increased in human lung adenocarcinoma and melanoma, and its levels were positively correlated with PD-L1 as well as its transcription factors IRF1 and HIF-2α. Collectively, our study identifies what we believe to be a previously unknown deubiquitinase, ATXN3, as a positive regulator for PD-L1 transcription and provides a rationale for targeting ATXN3 to sensitize checkpoint blockade antitumor immunotherapy.
Subject(s)
Lung Neoplasms , Tumor Escape , Humans , Animals , Mice , Tumor Escape/genetics , B7-H1 Antigen , Transcription Factors , Immunotherapy , Lung Neoplasms/pathology , Hypoxia , Deubiquitinating Enzymes , Basic Helix-Loop-Helix Transcription Factors , Cell Line, Tumor , Tumor Microenvironment , Ataxin-3 , Repressor ProteinsABSTRACT
The ubiquitin-specific peptidase Ataxin-3 (ATXN3) has emerged as a potential oncogene in a variety of human cancers. However, the molecular mechanisms underlying how ATXN3 achieves its tumorigenic functions remain largely undefined. Herein, we report that targeted deletion of the ATXN3 gene in cancer cells by the CRISPR-Cas9 system resulted in decreased protein expression of Yes-associated protein 1 (YAP1) without altering its mRNA transcription. Interestingly, genetic ATXN3 suppression selectively inhibited the expression levels of YAP1 target genes including the connective tissue growth factor (Ctgf) and cysteine-rich angiogenic inducer 61 (Cyr61), both of which have important functions in cell adhesion, migration, proliferation and angiogenesis. Consequently, ATXN3 suppression resulted in reduced cancer cell growth and migration, which can also be largely rescued by YAP1 reconstitution. At the molecular level, ATNX3 interacts with the WW domains of YAP1 to protect YAP1 from ubiquitination-mediated degradation. Immunohistology analysis revealed a strong positive correlation between ATXN3 and YAP1 protein expression in human breast and pancreatic cancers. Collectively, our study defines ATXN3 as a previously unknown YAP1 deubiquitinase in tumorigenesis and provides a rationale for ATXN3 targeting in antitumor chemotherapy.
ABSTRACT
[This corrects the article DOI: 10.3389/fnmol.2023.1182005.].
ABSTRACT
Objective: This study aims to explore whether interferon-induced transmembrane protein 3 (IFITM3) is involved in recombinant human brain natriuretic peptide (rhBNP)-mediated effects on sepsis-induced cognitive dysfunction in mice. Methods: The cellular localization and expression level of IFITM3 in the hippocampus were detected. The IFITM3 overexpression was achieved using an intracranial stereotactic system to inject an adeno-associated virus into the hippocampal CA1 region of mice. Field experiments, an elevated plus maze, and conditioned fear memory tests assessed the cognitive impairment in rhBNP-treated septic mice. Finally, in the hippocampus of septic mice, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining and Immunoblot were used to detect changes in the protein expression of cleaved Caspase-8 and cleaved Caspase-3 in apoptosis-related pathways, and toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) p65 in inflammatory pathways. Results: Fourteen days after cecal ligation and puncture (CLP) surgery, IFITM3 localized in the plasma membrane and cytoplasm of the astrocytes in the hippocampus of septic mice, partially attached to the perivascular and neuronal surfaces, but not expressed in the microglia. The expression of IFITM3 was increased in the astrocytes and neurons in the hippocampus of septic mice, which was selectively inhibited by the administration of rhBNP. Overexpression of IFITM3 resulted in elevated anxiety levels and long-term learning and memory dysfunction, completely abolished the therapeutic effect of rhBNP on cognitive impairment in septic mice, and induced an increase in the number of neuronal apoptosis in the hippocampal CA1 region. The expression levels of cleaved Caspase-3 and cleaved Caspase-8 proteins were significantly increased in the hippocampus, but the expression levels of TLR4 and NF-κB p65 were not increased. Conclusion: The activation of IFITM3 may be a potential new target for treating sepsis-associated encephalopathy (SAE), and it may be one of the key anti-apoptotic mechanisms in rhBNP exerting its therapeutic effect, providing new insight into the clinical treatment of SAE patients.
ABSTRACT
Integrins plays critical roles in connecting the extracellular matrix and actin skeleton for cell adhesion, migration, signal transduction, and gene transcription, which upregulation is involved in cancer stemness and metastasis. However, the molecular mechanisms underlying how integrins are upregulated in cancer stem cells (CSCs) remain as a biomedical mystery. Herein, we show that the death from cancer signature gene USP22 is essential to maintain the stemness of breast cancer cells through promoting the transcription of a group of integrin family members in particular integrin ß1 (ITGB1). Both genetic and pharmacological USP22 inhibition largely impaired breast cancer stem cell self-renewal and prevented their metastasis. Integrin ß1 reconstitution partially rescued USP22-null breast cancer stemness and their metastasis. At the molecular level, USP22 functions as a bona fide deubiquitinase to protect the proteasomal degradation of the forkhead box M1 (FoxM1), a transcription factor for tumoral ITGB1 gene transcription. Importantly unbiased analysis of the TCGA database revealed a strong positive correlation between the death from cancer signature gene ubiquitin-specific peptidase 22 (USP22) and ITGB1, both of which are critical for cancer stemness, in more than 90% of human cancer types, implying that USP22 functions as a key factor to maintain stemness for a broad spectrum of human cancer types possibly through regulating ITGB1. To support this notion, immunohistochemistry staining detected a positive correlation among USP22, FoxM1 and integrin ß1 in human breast cancers. Collectively, our study identifies the USP22-FoxM1-integrin ß1 signaling axis critical for cancer stemness and offers a potential target for antitumor therapy.
ABSTRACT
Defects in endoplasmic reticulum (ER) proteostasis have been linked to diseases in multiple organ systems. Here we examined the impact of perturbation of ER proteostasis in mice bearing thyrocyte-specific knockout of either HRD1 (to disable ER-associated protein degradation [ERAD]) or ATG7 (to disable autophagy) in the absence or presence of heterozygous expression of misfolded mutant thyroglobulin (the most highly expressed thyroid gene product, synthesized in the ER). Misfolding-inducing thyroglobulin mutations are common in humans but are said to yield only autosomal-recessive disease - perhaps because misfolded thyroglobulin protein might undergo disposal by ERAD or ER macroautophagy. We find that as single defects, neither ERAD, nor autophagy, nor heterozygous thyroglobulin misfolding altered circulating thyroxine levels, and neither defective ERAD nor defective autophagy caused any gross morphological change in an otherwise WT thyroid gland. However, heterozygous expression of misfolded thyroglobulin itself triggered significant ER stress and individual thyrocyte death while maintaining integrity of the surrounding thyroid epithelium. In this context, deficiency of ERAD (but not autophagy) resulted in patchy whole-follicle death with follicular collapse and degeneration, accompanied by infiltration of bone marrow-derived macrophages. Perturbation of thyrocyte ER proteostasis is thus a risk factor for both cell death and follicular demise.
Subject(s)
Thyroglobulin , Thyroid Gland , Humans , Animals , Mice , Thyroglobulin/genetics , Proteostasis , Autophagy , Endoplasmic ReticulumABSTRACT
P23, historically known as a heat shock protein 90 (HSP90) co-chaperone, exerts some of its critical functions in an HSP90-independent manner, particularly when it translocates into the nucleus. The molecular nature underlying how this HSP90-independent p23 function is achieved remains as a biological mystery. Here, we found that p23 is a previously unidentified transcription factor of COX-2, and its nuclear localization predicts the poor clinical outcomes. Intratumor succinate promotes p23 succinylation at K7, K33, and K79, which drives its nuclear translocation for COX-2 transcription and consequently fascinates tumor growth. We then identified M16 as a potent p23 succinylation inhibitor from 1.6 million compounds through a combined virtual and biological screening. M16 inhibited p23 succinylation and nuclear translocation, attenuated COX-2 transcription in a p23-dependent manner, and markedly suppressed tumor growth. Therefore, our study defines p23 as a succinate-activated transcription factor in tumor progression and provides a rationale for inhibiting p23 succinylation as an anticancer chemotherapy.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Humans , Succinic Acid , Transcription Factors/genetics , Cyclooxygenase 2/genetics , Pyridinolcarbamate , Carcinogenesis/genetics , Cell Transformation, Neoplastic , Succinates , Adenocarcinoma of Lung/genetics , Molecular Chaperones/genetics , HSP90 Heat-Shock Proteins/genetics , Lung Neoplasms/geneticsABSTRACT
Kidney damage due to ischemia or rejection results in the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) lumen, a condition known as "ER stress." Inositol-requiring enzyme 1α (IRE1α), the first ER stress sensor found, is a type I transmembrane protein with kinase and endoribonuclease activity. On activation, IRE1α nonconventionally splices an intron from unspliced X-box-binding protein 1 (XBP1) mRNA to produce XBP1s mRNA that encodes the transcription factor, XBP1s, for the expression of genes encoding proteins that mediate the unfolded protein response. The unfolded protein response promotes the functional fidelity of ER and is required for secretory cells to sustain protein folding and secretory capability. Prolonged ER stress can lead to apoptosis, which may result in detrimental repercussions to organ health and has been implicated in the pathogenesis and progression of kidney diseases. The IRE1α-XBP1 signaling acts as a major arm of unfolded protein response and is involved in regulating autophagy, cell differentiation, and cell death. IRE1α also interacts with activator protein-1 and nuclear factor-κB pathways to regulate inflammatory responses. Studies using transgenic mouse models highlight that the roles of IRE1α differ depending on cell type and disease setting. This review covers these cell-specific roles of IRE1α signaling and the potential for therapeutic targeting of this pathway in the context of ischemia and rejection affecting the kidneys.
Subject(s)
Endoribonucleases , Protein Serine-Threonine Kinases , Animals , Endoplasmic Reticulum Stress/genetics , Endoribonucleases/genetics , Endoribonucleases/metabolism , Graft Rejection , Inositol/metabolism , Kidney/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Unfolded Protein Response , HumansABSTRACT
T regulatory cells (Tregs) are a potential therapeutic target in many autoimmune diseases including rheumatoid arthritis (RA). The mechanisms responsible for the maintenance of Tregs in chronic inflammatory conditions such as RA are poorly understood. We employed our mouse model of RA in which, the following deletion of Flice-like inhibitory protein in CD11c+ cells, CD11c-FLIP-KO (HUPO) mice develop spontaneous, progressive, erosive arthritis, with reduced Tregs, and the adoptive transfer of Tregs ameliorates the arthritis. HUPO thymic Treg development was normal, but peripheral of Treg Foxp3 was diminished mediated by reduction of dendritic cells and interleukin-2 (IL-2). During chronic inflammatory arthritis Tregs fail to maintain Foxp3, leading to non-apoptotic cell death and conversion to CD4+CD25+Foxp3- cells. Treatment with IL-2 increased Tregs and ameliorated the arthritis. In summary, reduced dendritic cells and IL-2 in the milieu of chronic inflammation, contribute to Treg instability, promoting HUPO arthritis progression, and suggesting a therapeutic approach in RA.
ABSTRACT
Endoplasmic reticulum (ER)-associated degradation (ERAD) and ER-phagy are two principal degradative mechanisms for ER proteins and aggregates, respectively; however, the crosstalk between these two pathways under physiological settings remains unexplored. Using adipocytes as a model system, here we report that SEL1L-HRD1 protein complex of ERAD degrades misfolded ER proteins and limits ER-phagy and that, only when SEL1L-HRD1 ERAD is impaired, the ER becomes fragmented and cleared by ER-phagy. When both are compromised, ER fragments containing misfolded proteins spatially coalesce into a distinct architecture termed Coalescence of ER Fragments (CERFs), consisted of lipoprotein lipase (LPL, a key lipolytic enzyme and an endogenous SEL1L-HRD1 substrate) and certain ER chaperones. CERFs enlarge and become increasingly insoluble with age. Finally, we reconstitute the CERFs through LPL and BiP phase separation in vitro, a process influenced by both redox environment and C-terminal tryptophan loop of LPL. Hence, our findings demonstrate a sequence of events centered around SEL1L-HRD1 ERAD to dispose of misfolded proteins in the ER of adipocytes, highlighting the profound cellular adaptability to misfolded proteins in the ER in vivo.
Subject(s)
Proteins , Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , Proteins/metabolism , Endoplasmic Reticulum-Associated Degradation , Endoplasmic Reticulum/metabolism , Adipocytes/metabolismABSTRACT
Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including convalescent COVID-19 and sero-negative controls. Flow cytometry analyses revealed reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells within the patients who have recovered from severe COVID-19. sc-RNA seq analysis identifies seven heterogeneous clusters of monocytes and nine Treg clusters featuring distinct molecular signatures in association with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocytes and Tregs expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters featuring S100 family genes: one monocyte cluster of S100A8 & A9 coupled with high HLA-I and another cluster of S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, as well as a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-lived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (≥ 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.