MBTD1 preserves adult hematopoietic stem cell pool size and function.

Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a.

Near full-automation of COPMAN using a LabDroid enables high-throughput and sensitive detection of SARS-CoV-2 RNA in wastewater as a leading indicator.

Wastewater-based epidemiology (WBE) is a promising tool to efficiently monitor COVID-19 prevalence in a community. For WBE community surveillance, automation of the viral RNA detection process is ideal. In the present study, we achieved near full-automation of a previously established method, COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater), which was then applied to detect SARS-CoV-2 in wastewater for half a year. The automation line employed the Maholo LabDroid and an automated-pipetting device to achieve a high-throughput sample-processing capability of 576 samples per week. SARS-CoV-2 RNA was quantified with the automated COPMAN using samples collected from two wastewater treatment plants in the Sagami River basin in Japan between 1 November 2021 and 24 May 2022, when the numbers of daily reported COVID-19 cases ranged from 0 to 130.3 per 100,000 inhabitants. The automated COPMAN detected SARS-CoV-2 RNA from 81 out of 132 samples at concentrations of up to 2.8 × 105 copies/L. These concentrations showed direct correlations with subsequently reported clinical cases (5-13 days later), as determined by Pearson's and Spearman's cross-correlation analyses. To compare the results, we also conducted testing with the EPISENS-S (Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids, Ando et al., 2022), a previously reported detection method. SARS-CoV-2 RNA detected with EPISENS-S correlated with clinical cases only when using Spearman's method. Our automated COPMAN was shown to be an efficient method for timely and large-scale monitoring of viral RNA, making WBE more feasible for community surveillance.

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.

Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in initiation of lysophagy.

Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.

ARHGAP1 Transported with Influenza Viral Genome Ensures Integrity of Viral Particle Surface through Efficient Budozone Formation.

Influenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival. IMPORTANCE The endocytic transport of the influenza viral genome triggers the clustering of viral membrane proteins at the plasma membrane to form the viral budozone. However, host factors that promote viral budozone formation in concert with viral genome transport have not been identified. Here, we found that ARHGAP1, a negative regulator of the Rho family protein, is transported with the viral genome and stabilizes actin filaments to promote budozone formation. We have shown that ARHGAP1-mediated efficient formation of viral budozone was crucial for the clustering of viral HA protein to the progeny viral particles. The clustering of HA proteins on the virions is responsible for the structural integrity of the viral particles, which promotes viral stability and viral immune evasion. This study highlights the molecular mechanism that works in concert with viral genome packaging to ensure the structural integrity of viral particles.

ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

ß-adrenergic receptor signaling evokes the PKA-ASK axis in mature brown adipocytes.

Boosting energy expenditure by harnessing the activity of brown adipocytes is a promising strategy for combatting the global epidemic of obesity. Many studies have revealed that the ß3-adrenergic receptor agonist is a potent activator of brown adipocytes, even in humans, and PKA and p38 MAPK have been demonstrated for regulating the transcription of a wide range of critical genes such as Ucp1. We previously revealed that the PKA-ASK1-p38 axis is activated in immature brown adipocytes and contributes to functional maturation. However, the downstream mechanisms of PKA that initiate the p38 MAPK cascade are still mostly unknown in mature brown adipocytes. Here, we identified the ASK family as a crucial signaling molecule bridging PKA and MAPK in mature brown adipocytes. Mechanistically, the phosphorylation of ASK1 at threonine 99 and serine 993 is critical in PKA-dependent ASK1 activation. Additionally, PKA also activates ASK2, which contributes to MAPK regulation. These lines of evidence provide new details for tailoring a ßAR-dependent brown adipocyte activation strategy.

Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics.

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.

ERdj8 governs the size of autophagosomes during the formation process.

In macroautophagy, membrane structures called autophagosomes engulf substrates and deliver them for lysosomal degradation. Autophagosomes enwrap a variety of targets with diverse sizes, from portions of cytosol to larger organelles. However, the mechanism by which autophagosome size is controlled remains elusive. We characterized a novel ER membrane protein, ERdj8, in mammalian cells. ERdj8 localizes to a meshwork-like ER subdomain along with phosphatidylinositol synthase (PIS) and autophagy-related (Atg) proteins. ERdj8 overexpression extended the size of the autophagosome through its DnaJ and TRX domains. ERdj8 ablation resulted in a defect in engulfing larger targets. C. elegans, in which the ERdj8 orthologue dnj-8 was knocked down, could perform autophagy on smaller mitochondria derived from the paternal lineage but not the somatic mitochondria. Thus, ERdj8 may play a critical role in autophagosome formation by providing the capacity to target substrates of diverse sizes for degradation.

Promotion of the Warburg effect is associated with poor benefit from adjuvant chemotherapy in colorectal cancer.

Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5-fluorouracil-based adjuvant therapy. In addition, the efficacy of 5-fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5-fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5-fluorouracil adjuvant chemotherapy.

Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells.

BACKGROUND: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. METHODS: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. RESULTS: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. CONCLUSION: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.

Intrinsically Disordered Protein TEX264 Mediates ER-phagy.

Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.

Calpain-10 regulates actin dynamics by proteolysis of microtubule-associated protein 1B.

Calpain-10 (CAPN10) is the calpain family protease identified as the first candidate susceptibility gene for type 2 diabetes mellitus (T2DM). However, the detailed molecular mechanism has not yet been elucidated. Here we report that CAPN10 processes microtubule associated protein 1 (MAP1) family proteins into heavy and light chains and regulates their binding activities to microtubules and actin filaments. Immunofluorescent analysis of Capn10-/- mouse embryonic fibroblasts shows that MAP1B, a member of the MAP1 family of proteins, is localized at actin filaments rather than at microtubules. Furthermore, fluorescence recovery after photo-bleaching analysis shows that calpain-10 regulates actin dynamics via MAP1B cleavage. Moreover, in pancreatic islets from CAPN10 knockout mice, insulin secretion was significantly increased both at the high and low glucose levels. These findings indicate that deficiency of calpain-10 expression may affect insulin secretion by abnormal actin reorganization, coordination and dynamics through MAP1 family processing.

Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

ß-TrCP-dependent degradation of ASK1 suppresses the induction of the apoptotic response by oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a key player in the homeostatic response of many organisms. Of the many functions of ASK1, it is most well-known for its ability to induce canonical caspase 3-dependent apoptosis through the MAPK pathways in response to reactive oxygen species (ROS). As ASK1 is a regulator of apoptosis, its proper regulation is critical for the well-being of an organism. To date, several E3 ubiquitin ligases have been identified that are capable of degrading ASK1, signifying the importance of maintaining ASK1 expression levels during stress responses. ASK1 protein regulation under unstimulated conditions, however, is still largely unknown. Using tandem mass spectrometry, we have identified beta-transducin repeat containing protein (ß-TrCP), an E3 ubiquitin ligase, as a novel interacting partner of ASK1 that is capable of ubiquitinating and subsequently degrading ASK1 through the ubiquitin-proteasome system (UPS). This interaction requires the seven WD domains of ß-TrCP and the C-terminus of ASK1. By silencing the ß-TrCP genes, we observed a significant increase in caspase 3 activity in response to oxidative stress, which could subsequently be suppressed by silencing ASK1. These findings suggest that ß-TrCP is capable of suppressing oxidative stress-induced caspase 3-dependent apoptosis through suppression of ASK1, assisting in the organism's ability to maintain homeostasis in an unstable environment.

ZFP36L2 is a cell cycle-regulated CCCH protein necessary for DNA lesion-induced S-phase arrest.

ZFP36L2 promotes the destruction of AU-rich element-containing transcripts, while its regulation and functional significance in cell cycle control are scarcely identified. We show that ZFP36L2 is a cell cycle-regulated CCCH protein, the abundance of which is regulated post-translationally at the respective stages of the cell cycle. Indeed, ZFP36L2 protein was eliminated after release from M phase, and ZYG11B-based E3 ligase plays a role in its polyubiquitination in interphase. Although ZFP36L2 is dispensable for normal cell cycle progression, we found that endogenous ZFP36L2 played a key role in cisplatin-induced S-phase arrest, a process in which the suppression of G1/S cyclins is necessary. The accumulation of ZFP36L2 was stimulated under DNA replication stresses and altered interactions with a subset of RNA-binding proteins. Notably, silencing endogenous ZFP36L2 led to impaired cell viability in the presence of cisplatin-induced DNA lesions. Thus, we propose that ZFP36L2 is a key protein that controls S-phase progression in the case of genome instability.

Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells.

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.

TRIM48 Promotes ASK1 Activation and Cell Death through Ubiquitination-Dependent Degradation of the ASK1-Negative Regulator PRMT1.

Apoptosis signal-regulating kinase 1 (ASK1) is an oxidative stress-responsive kinase that is regulated by various interacting molecules and post-translational modifications. However, how these molecules and modifications cooperatively regulate ASK1 activity remains largely unknown. Here, we showed that tripartite motif 48 (TRIM48) orchestrates the regulation of oxidative stress-induced ASK1 activation. A pull-down screen identified a TRIM48-interacting partner, protein arginine methyltransferase 1 (PRMT1), which negatively regulates ASK1 activation by enhancing its interaction with thioredoxin (Trx), another ASK1-negative regulator. TRIM48 facilitates ASK1 activation by promoting K48-linked polyubiquitination and degradation of PRMT1. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death, whereas forced expression promoted cancer cell death in mouse xenograft model. These results indicate that TRIM48 facilitates oxidative stress-induced ASK1 activation and cell death through ubiquitination-dependent degradation of PRMT1. This study provides a cell death mechanism fine-tuned by the crosstalk between enzymes that engage various types of post-translational modifications.