Crystal Structure of the Human Cannabinoid Receptor CB2.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.

The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

Dysregulated Lung Commensal Bacteria Drive Interleukin-17B Production to Promote Pulmonary Fibrosis through Their Outer Membrane Vesicles.

Idiopathic pulmonary fibrosis (IPF) is a severe form of lung fibrosis with a high mortality rate. However, the etiology of IPF remains unknown. Here, we report that alterations in lung microbiota critically promote pulmonary fibrosis pathogenesis. We found that lung microbiota was dysregulated, and the dysregulated microbiota in turn induced production of interleukin-17B (IL-17B) during bleomycin-induced mouse lung fibrosis. Either lung-microbiota depletion or IL-17B deficiency ameliorated the disease progression. IL-17B cooperated with tumor necrosis factor-α to induce expression of neutrophil-recruiting genes and T helper 17 (Th17)-cell-promoting genes. Three pulmonary commensal microbes, which belong to the genera Bacteroides and Prevotella, were identified to promote fibrotic pathogenesis through IL-17R signaling. We further defined that the outer membrane vesicles (OMVs) that were derived from the identified commensal microbes induced IL-17B production through Toll-like receptor-Myd88 adaptor signaling. Together our data demonstrate that specific pulmonary symbiotic commensals can promote lung fibrosis by regulating a profibrotic inflammatory cytokine network.

Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos.

5-methylcytosine is a major epigenetic modification that is sometimes called "the fifth nucleotide." However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo's methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.

Valine tRNA levels and availability regulate complex I assembly in leukaemia.

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.

MPEK: a multitask deep learning framework based on pretrained language models for enzymatic reaction kinetic parameters prediction.

Enzymatic reaction kinetics are central in analyzing enzymatic reaction mechanisms and target-enzyme optimization, and thus in biomanufacturing and other industries. The enzyme turnover number (kcat) and Michaelis constant (Km), key kinetic parameters for measuring enzyme catalytic efficiency, are crucial for analyzing enzymatic reaction mechanisms and the directed evolution of target enzymes. Experimental determination of kcat and Km is costly in terms of time, labor, and cost. To consider the intrinsic connection between kcat and Km and further improve the prediction performance, we propose a universal pretrained multitask deep learning model, MPEK, to predict these parameters simultaneously while considering pH, temperature, and organismal information. Through testing on the same kcat and Km test datasets, MPEK demonstrated superior prediction performance over the previous models. Specifically, MPEK achieved the Pearson coefficient of 0.808 for predicting kcat, improving ca. 14.6% and 7.6% compared to the DLKcat and UniKP models, and it achieved the Pearson coefficient of 0.777 for predicting Km, improving ca. 34.9% and 53.3% compared to the Kroll_model and UniKP models. More importantly, MPEK was able to reveal enzyme promiscuity and was sensitive to slight changes in the mutant enzyme sequence. In addition, in three case studies, it was shown that MPEK has the potential for assisted enzyme mining and directed evolution. To facilitate in silico evaluation of enzyme catalytic efficiency, we have established a web server implementing this model, which can be accessed at http://mathtc.nscc-tj.cn/mpek.

Hepatitis B virus RNAs co-opt ELAVL1 for stabilization and CRM1-dependent nuclear export.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.

Construction of a human cell landscape at single-cell level.

Single-cell analysis is a valuable tool for dissecting cellular heterogeneity in complex systems1. However, a comprehensive single-cell atlas has not been achieved for humans. Here we use single-cell mRNA sequencing to determine the cell-type composition of all major human organs and construct a scheme for the human cell landscape (HCL). We have uncovered a single-cell hierarchy for many tissues that have not been well characterized. We established a 'single-cell HCL analysis' pipeline that helps to define human cell identity. Finally, we performed a single-cell comparative analysis of landscapes from human and mouse to identify conserved genetic networks. We found that stem and progenitor cells exhibit strong transcriptomic stochasticity, whereas differentiated cells are more distinct. Our results provide a useful resource for the study of human biology.

MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells.

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

Fine-Regional Role of the Claustrum in Anxiety and Higher Sensitivity to Cocaine in Adolescent Cocaine-Exposed Male Mice during Adulthood.

Adolescent cocaine exposure (ACE) induces anxiety and higher sensitivity to substances abuse during adulthood. Here, we show that the claustrum is crucial for controlling these psychiatric problems in male mice. In anxiety-like behavioral tests, the CaMKII-positive neurons in the median portion of the claustrum (MClaustrum) were triggered, and local suppression of these neurons reduced the anxiety-like behavior in ACE mice during adulthood. In contrast, the CaMKII-positive neurons in the anterior portion of the claustrum (AClaustrum) were more activated in response to subthreshold dose of cocaine induced conditioned place preference (CPP), and local suppression of these neurons blocked the acquisition of cocaine CPP in ACE mice during adulthood. Our findings for the first time identified the fine-regional role of the claustrum in regulating the anxiety and susceptibility to cocaine in ACE mice during adulthood, extending our understanding of the claustrum in substance use disorder.

Crosstalk of MAP3K1 and EGFR signaling mediates gene-environment interactions that block developmental tissue closure.

Aberrant regulation of signal transduction pathways can adversely derail biological processes for tissue development. One such process is the embryonic eyelid closure that is dependent on the mitogen-activated protein kinase kinase kinase 1 (MAP3K1). Map3k1 KO in mice results in defective eyelid closure and an autosomal recessive eye-open at birth phenotype. We have shown that in utero exposure to dioxin, a persistent environmental toxicant, induces the same eye defect in Map3k1+/- heterozygous but not WT pups. Here, we explore the mechanisms of the Map3k1 (gene) and dioxin (environment) interactions (GxE) underlying defective eyelid closure. We show that, acting through the aryl hydrocarbon receptor, dioxin activates epidermal growth factor receptor signaling, which in turn depresses MAP3K1-dependent Jun N-terminal kinase (JNK) activity. The dioxin-mediated JNK repression is moderate but is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1+/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1+/- pups, whereas knockout of Jnk1 and S1pr that encodes the sphigosin-1-phosphate (S1P) receptors upstream of the MAP3K1-JNK pathway potentiates the dioxin toxicity. Our novel findings show that the crosstalk of aryl hydrocarbon receptor, epidermal growth factor receptor, and S1P-MAP3K1-JNK pathways determines the outcome of dioxin exposure. Thus, gene mutations targeting these pathways are potential risk factors for the toxicity of environmental chemicals.

USP7 deubiquitinates epigenetic reader ZMYND8 to promote breast cancer cell migration and invasion.

The ubiquitin-proteasome system (UPS), which involves E3 ligases and deubiquitinases (DUBs), is critical for protein homeostasis. The epigenetic reader ZMYND8 (zinc finger MYND-type containing 8) has emerged as an oncoprotein, and its protein levels are elevated in various types of cancer, including breast cancer. However, the mechanism by which ZMYND8 protein levels are increased in cancer remains elusive. Although ZMYND8 has been reported to be regulated by the E3 ligase FBXW7, it is still unknown whether ZMYND8 could be modulated by DUBs. Here, we identified USP7 (ubiquitin carboxyl-terminal hydrolase 7) as a bona fide DUB for ZMYND8. Mechanically, USP7 directly binds to the PBP (PHD-BRD-PWWP) domain of ZMYND8 via its TRAF (tumor necrosis factor receptor-associated factor) domain and UBL (ubiquitin-like) domain and removes F-box and WD repeat domain containing 7 (FBXW7)-catalyzed poly-ubiquitin chains on lysine residue 1034 (K1034) within ZMYND8, thereby stabilizing ZMYND8 and stimulating the transcription of ZMYND8 target genes ZEB1 (zinc finger E-box binding homeobox 1) and VEGFA (Vascular Endothelial Growth Factor A). Consequently, USP7 enhances the capacity of breast cancer cells for migration and invasion through antagonizing FBXW7-mediated ZMYND8 degradation. Importantly, the protein levels of USP7 positively correlates with those of ZMYND8 in breast cancer tissues. These findings delineate an important layer of migration and invasion regulation by the USP7-ZMYND8 axis in breast cancer cells.

Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9-mediated inhibition of N6-methyladenosine demethylase FTO.

Porcine reproductive and respiratory syndrome virus (PRRSV), a highly infectious virus, causes severe losses in the swine industry by regulating the inflammatory response, inducing tissue damage, suppressing the innate immune response, and promoting persistent infection in hosts. Interleukin-13 (IL-13) is a cytokine that plays a critical role in regulating immune responses and inflammation, particularly in immune-related disorders, certain types of cancer, and numerous bacterial and viral infections; however, the underlying mechanisms of IL-13 regulation during PRRSV infection are not well understood. In this study, we demonstrated that PRRSV infection elevates IL-13 levels in porcine alveolar macrophages. PRRSV enhances m6A-methylated RNA levels while reducing the expression of fat mass and obesity associated protein (FTO, an m6A demethylase), thereby augmenting IL-13 production. PRRSV nonstructural protein 9 (nsp9) was a key factor for this modulation. Furthermore, we found that the residues Asp567, Tyr586, Leu593, and Asp595 were essential for nsp9 to induce IL-13 production via attenuation of FTO expression. These insights delineate PRRSV nsp9's role in FTO-mediated IL-13 release, advancing our understanding of PRRSV's impact on host immune and inflammatory responses.

Identification of metal ion-binding sites in RNA structures using deep learning method.

Metal ion is an indispensable factor for the proper folding, structural stability and functioning of RNA molecules. However, it is very difficult for experimental methods to detect them in RNAs. With the increase of experimentally resolved RNA structures, it becomes possible to identify the metal ion-binding sites in RNA structures through in-silico methods. Here, we propose an approach called Metal3DRNA to identify the binding sites of the most common metal ions (Mg2+, Na+ and K+) in RNA structures by using a three-dimensional convolutional neural network model. The negative samples, screened out based on the analysis for binding surroundings of metal ions, are more like positive ones than the randomly selected ones, which are beneficial to a powerful predictor construction. The microenvironments of the spatial distributions of C, O, N and P atoms around a sample are extracted as features. Metal3DRNA shows a promising prediction power, generally surpassing the state-of-the-art methods FEATURE and MetalionRNA. Finally, utilizing the visualization method, we inspect the contributions of nucleotide atoms to the classification in several cases, which provides a visualization that helps to comprehend the model. The method will be helpful for RNA structure prediction and dynamics simulation study. Availability and implementation: The source code is available at https://github.com/ChunhuaLiLab/Metal3DRNA.

Benchmarking multi-platform sequencing technologies for human genome assembly.

Genome assembly is a computational technique that involves piecing together deoxyribonucleic acid (DNA) fragments generated by sequencing technologies to create a comprehensive and precise representation of the entire genome. Generating a high-quality human reference genome is a crucial prerequisite for comprehending human biology, and it is also vital for downstream genomic variation analysis. Many efforts have been made over the past few decades to create a complete and gapless reference genome for humans by using a diverse range of advanced sequencing technologies. Several available tools are aimed at enhancing the quality of haploid and diploid human genome assemblies, which include contig assembly, polishing of contig errors, scaffolding and variant phasing. Selecting the appropriate tools and technologies remains a daunting task despite several studies have investigated the pros and cons of different assembly strategies. The goal of this paper was to benchmark various strategies for human genome assembly by combining sequencing technologies and tools on two publicly available samples (NA12878 and NA24385) from Genome in a Bottle. We then compared their performances in terms of continuity, accuracy, completeness, variant calling and phasing. We observed that PacBio HiFi long-reads are the optimal choice for generating an assembly with low base errors. On the other hand, we were able to produce the most continuous contigs with Oxford Nanopore long-reads, but they may require further polishing to improve on quality. We recommend using short-reads rather than long-reads themselves to improve the base accuracy of contigs from Oxford Nanopore long-reads. Hi-C is the best choice for chromosome-level scaffolding because it can capture the longest-range DNA connectedness compared to 10× linked-reads and Bionano optical maps. However, a combination of multiple technologies can be used to further improve the quality and completeness of genome assembly. For diploid assembly, hifiasm is the best tool for human diploid genome assembly using PacBio HiFi and Hi-C data. Looking to the future, we expect that further advancements in human diploid assemblers will leverage the power of PacBio HiFi reads and other technologies with long-range DNA connectedness to enable the generation of high-quality, chromosome-level and haplotype-resolved human genome assemblies.

Regulation of PKR-dependent RNA translation inhibition by TRIM21 upon virus infection or other stress.

The host always employs various ways to defend against viral infection and spread. However, viruses have evolved their own effective strategies, such as inhibition of RNA translation of the antiviral effectors, to destroy the host's defense barriers. Protein synthesis, commonly controlled by the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), is a basic cellular biological process among all species. In response to viral infection, in addition to inducing the transcription of antiviral cytokines by innate immunity, infected cells also inhibit the RNA translation of antiviral factors by activating the protein kinase R (PKR)-eIF2α signaling pathway. Regulation of innate immunity has been well studied; however, regulation of the PKR-eIF2α signaling pathway remains unclear. In this study, we found that the E3 ligase TRIM21 negatively regulates the PKR-eIF2α signaling pathway. Mechanistically, TRIM21 interacts with the PKR phosphatase PP1α and promotes K6-linked polyubiquitination of PP1α. Ubiquitinated PP1α augments its interaction with PKR, causing PKR dephosphorylation and subsequent translational inhibition release. Furthermore, TRIM21 can constitutively restrict viral infection by reversing PKR-dependent translational inhibition of various previously known and unknown antiviral factors. Our study highlights a previously undiscovered role of TRIM21 in regulating translation, which will provide new insights into the host antiviral response and novel targets for the treatment of translation-associated diseases in the clinic.

Hepatitis B virus hijacks TSG101 to facilitate egress via multiple vesicle bodies.

Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.

Construction of a cross-species cell landscape at single-cell level.

Individual cells are basic units of life. Despite extensive efforts to characterize the cellular heterogeneity of different organisms, cross-species comparisons of landscape dynamics have not been achieved. Here, we applied single-cell RNA sequencing (scRNA-seq) to map organism-level cell landscapes at multiple life stages for mice, zebrafish and Drosophila. By integrating the comprehensive dataset of > 2.6 million single cells, we constructed a cross-species cell landscape and identified signatures and common pathways that changed throughout the life span. We identified structural inflammation and mitochondrial dysfunction as the most common hallmarks of organism aging, and found that pharmacological activation of mitochondrial metabolism alleviated aging phenotypes in mice. The cross-species cell landscape with other published datasets were stored in an integrated online portal-Cell Landscape. Our work provides a valuable resource for studying lineage development, maturation and aging.

How many cell types are there in nature? How do they change during the life cycle? These are two fundamental questions that researchers have been trying to understand in the area of biology. In this study, single-cell mRNA sequencing data were used to profile over 2.6 million individual cells from mice, zebrafish and Drosophila at different life stages, 1.3 million of which were newly collected. The comprehensive datasets allow investigators to construct a cross-species cell landscape that helps to reveal the conservation and diversity of cell taxonomies at genetic and regulatory levels. The resources in this study are assembled into a publicly available website at http://bis.zju.edu.cn/cellatlas/.

Enhanced Thermoelectric Properties of Ti2CO2-Bismuthene-Ti2CO2: Optimized Power Factor and Reduced Thermal Conductivity.

In this study, bismuthene was intercalated between bilayer Ti2CTx to induce significant modifications in its electronic and phonon structures, thereby enhancing its thermoelectric properties. First-principles calculations reveal that the insertion of bismuthene transforms the Ti2CO2 system from a semiconductor into a metal and optimizes the thermoelectric properties of bilayer Ti2CO2 by enhancing its power factor and reducing its lattice thermal conductivity. Under the first-principles calculation parameters used in this study, the ZT of the Ti2CO2 system increased from 0.12 to 0.55. Conversely, for metallic bilayer MXenes, the introduction of bismuthene led to a substantial decrease in ZT (from 0.53 to 0.11 in the Ti2C system and from 0.07 to 0.05 in the Ti2CCl2 system). This study investigates the physical mechanisms underlying the enhancement of thermoelectric properties from both electronic and phononic perspectives and provides theoretical insights into the development and application of MXene-based thermoelectric materials.