Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.

Immune checkpoint inhibition targeting the PD-1/PD-L1 pathway has become a powerful clinical strategy for treating cancer, but its efficacy is complicated by various resistance mechanisms. One of the reasons for the resistance is the internalization and recycling of PD-L1 itself upon antibody binding. The inhibition of lysosome-mediated degradation of PD-L1 is critical for preserving the amount of PD-L1 recycling back to the cell membrane. In this study, we find that Hsc70 promotes PD-L1 degradation through the endosome-lysosome pathway and reduces PD-L1 recycling to the cell membrane. This effect is dependent on Hsc70-PD-L1 binding which inhibits the CMTM6-PD-L1 interaction. We further identify an Hsp90α/ß inhibitor, AUY-922, which induces Hsc70 expression and PD-L1 lysosomal degradation. Either Hsc70 overexpression or AUY-922 treatment can reduce PD-L1 expression, inhibit tumor growth and promote anti-tumor immunity in female mice; AUY-922 can further enhance the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 treatment. Our study elucidates a molecular mechanism of Hsc70-mediated PD-L1 lysosomal degradation and provides a target and therapeutic strategies for tumor immunotherapy.

Mitophagy defect mediates the aging-associated hallmarks in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease manifested by premature aging and aging-related phenotypes, making it a disease model for aging. The cellular machinery mediating age-associated phenotypes in HGPS remains largely unknown, resulting in limited therapeutic targets for HGPS. In this study, we showed that mitophagy defects impaired mitochondrial function and contributed to cellular markers associated with aging in mesenchymal stem cells derived from HGPS patients (HGPS-MSCs). Mechanistically, we discovered that mitophagy affected the aging-associated phenotypes of HGPS-MSCs by inhibiting the STING-NF-ĸB pathway and the downstream transcription of senescence-associated secretory phenotypes (SASPs). Furthermore, by utilizing UMI-77, an effective mitophagy inducer, we showed that mitophagy induction alleviated aging-associated phenotypes in HGPS and naturally aged mice. Collectively, our results uncovered that mitophagy defects mediated the aging-associated markers in HGPS, highlighted the function of mitochondrial homeostasis in HGPS progression, and suggested mitophagy as an intervention target for HGPS and aging.

Novel mitophagy inducer alleviates lupus nephritis by reducing myeloid cell activation and autoantigen presentation.

Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE), but its mechanism of onset remains unclear. Since impaired mitophagy has been implicated in multiple organs in SLE, we hypothesized that mitophagy dysfunction is critical in the development of LN and that pharmacologically targeting mitophagy would ameliorate this disease. Therefore, lupus-prone MRL/MpJ-Faslpr (MRL/lpr) and NZBWF1/J mice were treated with a novel mitophagy inducer, UMI-77, during their onset of LN. This treatment effectively mitigated kidney inflammation and damage as assessed by histology and flow cytometry. Furthermore, dendritic cell (DC)-T-cell coculture assay indicated that UMI-77 treatment attenuated DC function that would drive T-cell proliferation but did not directly influence the potent T-cell proliferation in lupus mice. UMI-77 also restored mitochondrial function and attenuated proinflammatory phenotypes in lupus DCs. Adoptive transfer of DCs from MRL/lpr mice augmented serum anti-dsDNA IgG, urine protein and T-cell infiltration of the kidney in MRL/MpJ mice, which could be prevented by either treating lupus donors in vivo or lupus DCs directly with UMI-77. UMI-77 also restored mitochondrial function in myeloid cells from patients with LN in vitro as evidenced by increased ATP levels. Thus, enhancing mitophagy in SLE restrains autoimmunity and limits kidney inflammation for LN development. Hence, our findings suggest targeting mitophagy as a tangible pathway to treat LN.

ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade.

Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance mechanisms. Using an ICB-insensitive tumor model, here we discover cisplatin enhances the anti-tumor effect of PD-L1 blockade and upregulates the expression of Ariadne RBR E3 ubiquitin-protein ligase 1 (ARIH1) in tumors. Arih1 overexpression promotes cytotoxic T cell infiltration, inhibits tumor growth, and potentiates PD-L1 blockade. ARIH1 mediates ubiquitination and degradation of DNA-PKcs to trigger activation of the STING pathway, which is blocked by the phospho-mimetic mutant T68E/S213D of cGAS protein. Using a high-throughput drug screen, we further identify that ACY738, less cytotoxic than cisplatin, effectively upregulates ARIH1 and activates STING signaling, sensitizing tumors to PD-L1 blockade. Our findings delineate a mechanism that tumors mediate ICB resistance through the loss of ARIH1 and ARIH1-DNA-PKcs-STING signaling and indicate that activating ARIH1 is an effective strategy to improve the efficacy of cancer immunotherapy.

Spatially resolved proteomics via tissue expansion.

Spatially resolved proteomics is an emerging approach for mapping proteome heterogeneity of biological samples, however, it remains technically challenging due to the complexity of the tissue microsampling techniques and mass spectrometry analysis of nanoscale specimen volumes. Here, we describe a spatially resolved proteomics method based on the combination of tissue expansion with mass spectrometry-based proteomics, which we call Expansion Proteomics (ProteomEx). ProteomEx enables quantitative profiling of the spatial variability of the proteome in mammalian tissues at ~160 µm lateral resolution, equivalent to the tissue volume of 0.61 nL, using manual microsampling without the need for custom or special equipment. We validated and demonstrated the utility of ProteomEx for streamlined large-scale proteomics profiling of biological tissues including brain, liver, and breast cancer. We further applied ProteomEx for identifying proteins associated with Alzheimer's disease in a mouse model by comparative proteomic analysis of brain subregions.

SC75741, A Novel c-Abl Inhibitor, Promotes the Clearance of TDP25 Aggregates via ATG5-Dependent Autophagy Pathway.

Abnormal accumulation of TDP43-related mutant proteins in the cytoplasm causes amyotrophic lateral sclerosis (ALS). Herein, unbiased drug screening approaches showed that SC75741, a multi-target inhibitor, inhibited inflammation-induced aggregation by inhibiting NF-κB and also degraded already aggregated proteins by inhibiting c-Abl mediated autophagy-lysosomal pathway. We delineate the mechanism that SC75741 could markedly enhance TFEB nuclear translocation by an mTORC1-independent TFEB regulatory pathway. In addition, SC75741 enhanced the interaction between p62 with TDP25 and LC3C, thus promoting TDP25 degradation. Taken together, these findings show that SC75741 has beneficial neuroprotective effects in ALS. Our study elucidates that dual-targeted inhibition of c-Abl and NF-κB may be a potential treatment for TDP43 proteinopathies and ALS.

Mitophagy Regulates Neurodegenerative Diseases.

Mitochondria play an essential role in supplying energy for the health and survival of neurons. Mitophagy is a metabolic process that removes dysfunctional or redundant mitochondria. This process preserves mitochondrial health. However, defective mitophagy triggers the accumulation of damaged mitochondria, causing major neurodegenerative disorders. This review introduces molecular mechanisms and signaling pathways behind mitophagy regulation. Furthermore, we focus on the recent advances in understanding the potential role of mitophagy in the pathogenesis of major neurodegenerative diseases (Parkinson's, Alzheimer's, Huntington's, etc.) and aging. The findings will help identify the potential interventions of mitophagy regulation and treatment strategies of neurodegenerative diseases.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model.

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/ß signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/ß-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aß plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/ß-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation.

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

Targeting MCL1 to induce mitophagy is a potential therapeutic strategy for Alzheimer disease.

Mitochondrial dysfunction is associated with the occurrence of a variety of neurodegenerative diseases, especially Alzheimer disease (AD). As a mitochondrial quality control process, mitophagy is greatly inhibited in AD; increasing evidence shows that the induction of mitophagy is an effective therapeutic intervention strategy. However, the lack of more safe, effective, and clear mechanisms for mitophagy inducers has limited the clinical application. In recent studies, we have identified a small molecule compound, UMI-77, that can safely and effectively induce mitophagy. UMI-77 is an established BH3-mimetic for MCL1 and was developed to induce apoptosis in cancer cells. We found that UMI-77 can bind MCL1 and enhance its function as a mitophagy receptor protein, thus enhancing its interaction with LC3A to induce mitophagy. UMI-77 effectively improves the cognitive decline seen in an AD mouse model. Our findings shed light on the novel mechanisms of mitophagy, reveal that MCL1 is a mitophagy receptor that can be targeted to induce mitophagy, and identify MCL1 as a drug target for therapeutic intervention in AD.

Pharmacological targeting of MCL-1 promotes mitophagy and improves disease pathologies in an Alzheimer's disease mouse model.

There is increasing evidence that inducing neuronal mitophagy can be used as a therapeutic intervention for Alzheimer's disease. Here, we screen a library of 2024 FDA-approved drugs or drug candidates, revealing UMI-77 as an unexpected mitophagy activator. UMI-77 is an established BH3-mimetic for MCL-1 and was developed to induce apoptosis in cancer cells. We found that at sub-lethal doses, UMI-77 potently induces mitophagy, independent of apoptosis. Our mechanistic studies discovered that MCL-1 is a mitophagy receptor and directly binds to LC3A. Finally, we found that UMI-77 can induce mitophagy in vivo and that it effectively reverses molecular and behavioral phenotypes in the APP/PS1 mouse model of Alzheimer's disease. Our findings shed light on the mechanisms of mitophagy, reveal that MCL-1 is a mitophagy receptor that can be targeted to induce mitophagy, and identify MCL-1 as a drug target for therapeutic intervention in Alzheimer's disease.

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs.

Molecular evidence for the coordination of nitrogen and carbon metabolisms, revealed by a study on the transcriptional regulation of the agl3EFG operon that encodes a putative carbohydrate transporter in Streptomyces coelicolor.

In the agl3EFGXYZ operon (SCO7167-SCO7162, abbreviated as agl3 operon) of Streptomyces coelicolor M145, agl3EFG genes encode a putative ABC-type carbohydrate transporter. The transcription of this operon has been proved to be repressed by Agl3R (SCO7168), a neighboring GntR-family regulator, and this repression can be released by growth on poor carbon sources. Here in this study, we prove that the transcription of agl3 operon is also directly repressed by GlnR, a central regulator governing the nitrogen metabolism in S. coelicolor. The electrophoretic mobility shift assay (EMSA) employing the agl3 promoter and mixtures of purified recombinant GlnR and Agl3R indicates that GlnR and Agl3R bind to different DNA sequences within the promoter region of agl3 operon, which is further confirmed by the DNase I footprinting assay. As Agl3R and GlnR have been demonstrated to sense the extracellular carbon and nitrogen supplies, respectively, it is hypothesized that the transcription of agl3 operon is stringently governed by the availabilities of extracellular carbon and nitrogen sources. Consistent with the hypothesis, the agl3 operon is further found to be derepressed only under the condition of poor carbon and rich nitrogen supplies, when both regulators are inactivated. It is believed that activation of the expression of agl3 operon may facilitate the absorption of extracellular carbohydrates to balance the ratio of intracellular carbon to nitrogen.

Streptomyces yangpuensis sp. nov., isolated from soil.

A novel Gram-stain-positive strain with sandy aerial mycelium and golden yellow substrate mycelium, designated fd2-tbT, was isolated from a soil sample collected in Shanghai, China, and its taxonomic status was established by phylogenetic analysis. 16S rRNA gene sequence analysis showed that strain fd2-tbT belonged to the genus Streptomyces and was related to Streptomyces amritsarensis JCM 19660T (99.9 % 16S rRNA gene sequence similarity), Streptomyces flavotricini NBRC 12770T (99.9 %), Streptomyces polychromogenes NBRC 13072T (99.8 %), Streptomyces racemochromogenes NRRL B-5430T (99.7 %), Streptomyces globosus LMG 19896T (99.5 %), Streptomyces toxytricini NBRC 12823T (99.5 %) and Streptomyces katrae NBRC 13447T (99.3 %). The cell wall of strain fd2-tbT contained ll-diaminopimelic acid, and whole-cell sugars were identified as glucose and ribose. The menaquinones MK-9(H4), MK-9(H6) and MK-9(H8) were also detected. In addition, the polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, as well as five unidentified phospholipids, were detected. Major cellular fatty acids were identified as anteiso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and C16 : 0. DNA-DNA hybridization experiments showed that strain fd2-tbT exhibited 36.5 ± 0.6 %, 43.5 ± 2.0 %, 11.1 ± 1.3 %, 10.3 ± 3.1 %, 9.8 ± 1.9 %, 48.9 ± 3.9 % and 16.3 ± 1.7 % relatedness to S. amritsarensis JCM 40119660T, S. flavotricini NBRC 12770T, S. polychromogenes NBRC 13072T, S. racemochromogenes NRRL B-5430T, S. globosus LMG 19896T, S. toxytricini NBRC 12823T and S. katrae NBRC 13447T, respectively. Based on these analyses as well as some phenotypic differences, strain fd2-tbT is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces yangpuensis sp. nov. is proposed. The type strain is fd2-tbT ( = DSM 100336T = CGMCC 4.7256T).

A systematic study of the whole genome sequence of Amycolatopsis methanolica strain 239T provides an insight into its physiological and taxonomic properties which correlate with its position in the genus.

The complete genome of methanol-utilizing Amycolatopsis methanolica strain 239T was generated, revealing a single 7,237,391 nucleotide circular chromosome with 7074 annotated protein-coding sequences (CDSs). Comparative analyses against the complete genome sequences of Amycolatopsis japonica strain MG417-CF17T, Amycolatopsis mediterranei strain U32 and Amycolatopsis orientalis strain HCCB10007 revealed a broad spectrum of genomic structures, including various genome sizes, core/quasi-core/non-core configurations and different kinds of episomes. Although polyketide synthase gene clusters were absent from the A. methanolica genome, 12 gene clusters related to the biosynthesis of other specialized (secondary) metabolites were identified. Complete pathways attributable to the facultative methylotrophic physiology of A. methanolica strain 239T, including both the mdo/mscR encoded methanol oxidation and the hps/hpi encoded formaldehyde assimilation via the ribulose monophosphate cycle, were identified together with evidence that the latter might be the result of horizontal gene transfer. Phylogenetic analyses based on 16S rDNA or orthologues of AMETH_3452, a novel actinobacterial class-specific conserved gene against 62 or 18 Amycolatopsis type strains, respectively, revealed three major phyletic lineages, namely the mesophilic or moderately thermophilic A. orientalis subclade (AOS), the mesophilic Amycolatopsis taiwanensis subclade (ATS) and the thermophilic A. methanolica subclade (AMS). The distinct growth temperatures of members of the subclades correlated with corresponding genetic variations in their encoded compatible solutes. This study shows the value of integrating conventional taxonomic with whole genome sequence data.

Draft Genome Sequence of the Streptothricin-Producing Strain Streptomyces sp. fd2-tb.

Streptomyces sp. fd2-tb can produce streptothricin class antibiotics with broad antimicrobial spectra. To better understand the mechanism of streptothricin biosynthesis and to assess the capacity of this strain in secondary metabolism, we report the draft genome sequence of Streptomyces sp. strain fd2-tb.

Characterization of a new GlnR binding box in the promoter of amtB in Streptomyces coelicolor inferred a PhoP/GlnR competitive binding mechanism for transcriptional regulation of amtB.

The transcription of amtB in Streptomyces coelicolor has been proposed to be counter-regulated by GlnR (a global regulator for nitrogen metabolism) and PhoP (a global regulator for phosphate metabolism). However, the GlnR-protected region, which was deduced to be two 22-bp GlnR binding boxes (gTnAc-n6-GaAAc-n6-GtnAC-n6-GAAAc-n6, abbreviated as a1-b1 and a2-b2), was separated from the PhoP-protected region in the promoter of amtB, leaving the mechanism for this regulation undefined. In this study, another 22-bp GlnR binding box, which consisted of a3-site-n6-b3-site (a3-b3) overlapping with the PhoP-binding sequences, was identified in the promoter region of amtB by a DNase I footprinting assay. An electrophoretic mobility shift assay (EMSA) using purified recombinant GlnR and the synthetic amtB promoter fragments with the three GlnR binding boxes individually mutated demonstrated that every box was involved in GlnR binding in vitro. Further in vivo assays using the egfp reporter gene fused to various kinds of mutated promoter regions of amtB demonstrated that all of the three GlnR binding boxes were required for GlnR-mediated activation of amtB transcription under the nitrogen-limited condition. The results of EMSA using the amtB promoter with mixtures of recombinant His-tagged GlnR and Trx-His-S-tagged PhoP inferred that PhoP might compete against GlnR from binding at the a3-b3 site, attributable to the PhoP/GlnR counter-regulatory function subjected to further experimental proof.

Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism.

Amycolatopsis mediterranei is used for industry-scale production of rifamycin, which plays a vital role in antimycobacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10,236,715 base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9,228 protein-coding genes in the A. mediterranei genome shared the greatest number of orthologs with those of S. erythraea, it was unexpectedly followed by Streptomyces coelicolor rather than N. farcinica, indicating the distinct metabolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metabolite production are located. The rifamycin biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of rifamycin SV to B, revealed by comparing to the highly homologous cluster of the rifamycin B-producing strain S699 and further confirmed by genetic complementation. The genomic information of A. mediterranei demonstrates a metabolic network orchestrated not only for extensive utilization of various carbon sources and inorganic nitrogen compounds but also for effective funneling of metabolic intermediates into the secondary antibiotic synthesis process under the control of a seemingly complex regulatory mechanism.