The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

A multistage Sendai virus vaccine incorporating latency-associated antigens induces protection against acute and latent tuberculosis.

One-quarter of the world's population is infected with Mycobacterium tuberculosis (Mtb). After initial exposure, more immune-competent persons develop asymptomatic latent tuberculosis infection (LTBI) but not active diseases, creates an extensive reservoir at risk of developing active tuberculosis. Previously, we constructed a novel recombinant Sendai virus (SeV)-vectored vaccine encoding two dominant antigens of Mtb, which elicited immune protection against acute Mtb infection. In this study, nine Mtb latency-associated antigens were screened as potential supplementary vaccine candidate antigens, and three antigens (Rv2029c, Rv2028c, and Rv3126c) were selected based on their immune-therapeutic effect in mice, and their elevated immune responses in LTBI human populations. Then, a recombinant SeV-vectored vaccine, termed SeV986A, that expresses three latency-associated antigens and Ag85A was constructed. In murine models, the doses, titers, and inoculation sites of SeV986A were optimized, and its immunogenicity in BCG-primed and BCG-naive mice were determined. Enhanced immune protection against the Mtb challenge was shown in both acute-infection and latent-infection murine models. The expression levels of several T-cell exhaustion markers were significantly lower in the SeV986A-vaccinated group, suggesting that the expression of latency-associated antigens inhibited the T-cell exhaustion process in LTBI infection. Hence, the multistage quarter-antigenic SeV986A vaccine holds considerable promise as a novel post-exposure prophylaxis vaccine against tuberculosis.

Transcription factor Sp1 transcriptionally enhances GSDME expression for pyroptosis.

Gasdermin-E (GSDME), the executioner of pyroptosis when cleaved by caspase 3, plays a crucial role in tumor defense and the response to chemotherapy drugs in cells. So far, there are poorly known mechanisms for the expression regulation of GSDME during cell death. Here, we identify the transcription factor Sp1 (Specificity protein 1) as a positive regulator of GSDME-mediated pyroptosis. Sp1 directly interacts with the GSDME promoter at -36 ~ -28 site and promotes GSDME gene transcription. Further, Sp1 knockdown or inhibition suppresses GSDME expression, thus reducing chemotherapy drugs (topotecan, etoposide, doxorubicin, sorafinib and cisplatin) induced cell pyroptosis. The regulation process synergizes with STAT3 (Signal transducer and activator of transcription 3) activity and antagonizes with DNA methylation but barely affects GSDMD-mediated pyroptosis or TNF-induced necroptosis. Our current finding reveals a new regulating mechanism of GSDME expression, which may be a viable target for the intervention of GSDME-dependent inflammatory diseases and cancer therapy.

Enhanced neutralization of SARS-CoV-2 variant BA.2.86 and XBB sub-lineages by a tetravalent COVID-19 vaccine booster.

Emerging SARS-CoV-2 sub-lineages like XBB.1.5, XBB.1.16, EG.5, HK.3 (FLip), and XBB.2.3 and the variant BA.2.86 have recently been identified. Understanding the efficacy of current vaccines on these emerging variants is critical. We evaluate the serum neutralization activities of participants who received COVID-19 inactivated vaccine (CoronaVac), those who received the recently approved tetravalent protein vaccine (SCTV01E), or those who had contracted a breakthrough infection with BA.5/BF.7/XBB virus. Neutralization profiles against a broad panel of 30 sub-lineages reveal that BQ.1.1, CH.1.1, and all the XBB sub-lineages exhibit heightened resistance to neutralization compared to previous variants. However, despite their extra mutations, BA.2.86 and the emerging XBB sub-lineages do not demonstrate significantly increased resistance to neutralization over XBB.1.5. Encouragingly, the SCTV01E booster consistently induces higher neutralizing titers against all these variants than breakthrough infection does. Cellular immunity assays also show that the SCTV01E booster elicits a higher frequency of virus-specific memory B cells. Our findings support the development of multivalent vaccines to combat future variants.

MLKL deficiency alleviates neuroinflammation and motor deficits in the α-synuclein transgenic mouse model of Parkinson's disease.

Parkinson's disease (PD), one of the most devastating neurodegenerative brain disorders, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of α-synuclein aggregates. Currently, pharmacological interventions for PD remain inadequate. The cell necroptosis executor protein MLKL (Mixed-lineage kinase domain-like) is involved in various diseases, including inflammatory bowel disease and neurodegenerative diseases; however, its precise role in PD remains unclear. Here, we investigated the neuroprotective role of MLKL inhibition or ablation against primary neuronal cells and human iPSC-derived midbrain organoids induced by toxic α-Synuclein preformed fibrils (PFFs). Using a mouse model (Tg-Mlkl-/-) generated by crossbreeding the SNCA A53T synuclein transgenic mice with MLKL knockout (KO)mice, we assessed the impact of MLKL deficiency on the progression of Parkinsonian traits. Our findings demonstrate that Tg-Mlkl-/- mice exhibited a significant improvement in motor symptoms and reduced phosphorylated α-synuclein expression compared to the classic A53T transgenic mice. Furthermore, MLKL deficiency alleviated tyrosine hydroxylase (TH)-positive neuron loss and attenuated neuroinflammation by inhibiting the activation of microglia and astrocytes. Single-cell RNA-seq (scRNA-seq) analysis of the SN of Tg-Mlkl-/- mice revealed a unique cell type-specific transcriptome profile, including downregulated prostaglandin D synthase (PTGDS) expression, indicating reduced microglial cells and dampened neuron death. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing motor deficits in PD.

Correction: Wang et al. A VLP-Based Vaccine Displaying HBHA and MTP Antigens of Mycobacterium tuberculosis Induces Potentially Protective Immune Responses in M. tuberculosis H37Ra Infected Mice. Vaccines 2023, 11, 941.

Concerns were brought to the attention of the journal's editorial office after the paper was published [...].

Identification of disulfidptosis-related subtypes and development of a prognosis model based on stacking framework in renal clear cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor with an unsatisfactory prognosis. This study aims to identify the expression patterns of disulfidptosis-related genes (DRGs), develop a prognostic model, and predict immunological profiles. METHODS: First, we identified differentially expressed DRGs in TCGA-KIRC cohort and analyzed their mutational profiles, methylation levels, and interaction networks. Subsequently, we identified disulfidptosis-associated molecular subtypes and investigated their prognostic and immunological characteristics. Simultaneously, a disulfidptosis-related prognostic signature (DRPS) was developed using a two-stage stacking framework consisting of 5 machine learning models. The effect of DRPS on immune cell infiltration levels was explored using seven different algorithms, and the status and function of T cells for distinct risk-score groups were evaluated based on T cell exhaustion and dysfunction scores. Additionally, the study also examined differences in clinical characteristics and therapy efficacy between high- and low-risk groups. RESULTS: We found two disulfidptosis-associated clusters, one of which had a poor prognosis and was linked to high immune cell infiltration but impaired T cell function. DRPS showed excellent predictive performance in all four cohorts and could accurately identified disulfidptosis-related molecular subtypes. The DRPS-based risk score was positively associated with poor prognosis, malignant pathological features, high immune cell infiltration levels, and T cell exhaustion or dysfunction, and better respond to immunotherapy and targeted therapy. Additionally, we have identified a close association between ISG20 and disulfidptosis as well as tumor immunity. CONCLUSION: Our study identified distinct disulfidptosis-related subtypes in ccRCC patients, and constructed the highly accurate and robust DRPS based on an ensemble learning framework, which has critical reference value in clinical decision-making and individualized treatment. And this work also revealed ISG20 exhibits promising potential as a therapeutic target for ccRCC.

HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8 (HSPA8) as a new type of enzyme - which we name as 'amyloidase' - that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X1)φ(X3). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

A VLP-Based Vaccine Displaying HBHA and MTP Antigens of Mycobacterium tuberculosis Induces Protective Immune Responses in M. tuberculosis H37Ra Infected Mice.

Heparin-binding hemagglutinin (HBHA) and M. tuberculosis pili (MTP) are important antigens on the surface of Mycobacterium tuberculosis. To display these antigens effectively, the fusion protein HBHA-MTP with a molecular weight of 20 kD (L20) was inserted into the receptor-binding hemagglutinin (HA) fragment of influenza virus and was expressed along with matrix protein M1 in Sf9 insect cells to generate influenza virus-like particles (LV20 in short). The results showed that the insertion of L20 into the envelope of the influenza virus did not affect the self-assembly and morphology of LV20 VLPs. The expression of L20 was successfully verified by transmission electron microscopy. Importantly, it did not interfere with the immunogenicity reactivity of LV20 VLPs. We demonstrated that LV20 combined with the adjuvant composed of DDA and Poly I: C (DP) elicited significantly higher antigen-specific antibodies and CD4+/CD8+ T cell responses than PBS and BCG vaccination in mice. It suggests that the insect cell expression system is an excellent protein production system, and LV20 VLPs could be a novel tuberculosis vaccine candidate for further evaluation.

Systematic pan-cancer analysis identifies gasdermin B as an immunological and prognostic biomarker for kidney renal clear cell carcinoma.

Gasdermin (GSDM)-mediated cell lytic death plays an essential role in immunity and tumorigenesis. Despite the association of gasdermin B (GSDMB) with the tumorigenesis of various cancers, whether GSDMB functions as a prognostic biomarker in renal cell carcinoma remains poorly understood. Here, we explored the potential immunological functions and the prognostic value of GSDMB across multiple tumors with The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, including analyzing the relationship between GSDMB expression and prognosis, tumor-immune system interactions, immunomodulators, and immune cell infiltration of different tumors. Importantly, elevated expression of GSDMB is an essential factor for the poor prognosis of kidney renal clear cell carcinoma (KIRC) patients, suggesting that it might be helpful to predict a survival benefit from a clinical therapy regimen. Furthermore, GSDMB expression promoted the level of CD4+ T-cell infiltration of the tumors but is significantly negatively associated with immature dendritic cells (iDCs) in KIRC. Additionally, we identified TNFRSF25 and TNFSF14 as immunostimulators highly correlated with GSDMB expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that GSDMB and its interacting proteins might affect tumor growth through the serine metabolism pathway. Our current results demonstrate a promising therapeutic strategy targeting GSDMB and provide new insights into GSDMB as an immunological and prognostic biomarker for KIRC.

Structural insights into RNase J that plays an essential role in Mycobacterium tuberculosis RNA metabolism.

Ribonucleases (RNases) are responsible for RNA metabolism. RNase J, the core enzyme of the RNA degradosome, plays an essential role in global mRNA decay. Emerging evidence showed that the RNase J of Mycobacterium tuberculosis (Mtb-RNase J) could be an excellent target for treating Mtb infection. Here, crystal structures of Mtb-RNase J in apo-state and complex with the single-strand RNA reveal the conformational change upon RNA binding and hydrolysis. Mtb-RNase J forms an active homodimer through the interactions between the ß-CASP and the ß-lactamase domain. Knockout of RNase J slows the growth rate and changes the colony morphologies and cell length in Mycobacterium smegmatis, which is restored by RNase J complementation. Finally, RNA-seq analysis shows that the knockout strain significantly changes the expression levels of 49 genes in metabolic pathways. Thus, our current study explores the structural basis of Mtb-RNase J and might provide a promising candidate in pharmacological treatment for tuberculosis.

Structural analysis of the housecleaning nucleoside triphosphate pyrophosphohydrolase MazG from Mycobacterium tuberculosis.

The housecleaning enzyme of Mycobacterium tuberculosis (Mtb), MazG, is a nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) and can hydrolyze all canonical or non-canonical NTPs into NMPs and pyrophosphate. The Mycobacterium tuberculosis MazG (Mtb-MazG) contributes to antibiotic resistance in response to oxidative or nitrosative stress under dormancy, making it a promising target for treating TB in latent infection patients. However, the structural basis of Mtb-MazG is not clear. Here we describe the crystal structure of Mtb-MazG (1-185) at 2.7 Å resolution, composed of two similar folded spherical domains in tandem. Unlike other all-α NTP pyrophosphatases, Mtb-MazG has an N-terminal extra region composed of three α-helices and five ß-strands. The second domain is global, with five α-helices located in the N-terminal domain. Gel-filtration assay and SAXS analysis show that Mtb-MazG forms an enzyme-active dimer in solution. In addition, the metal ion Mg2+ is bound with four negative-charged residues Glu119, Glu122, Glu138, and Asp141. Different truncations and site-directed mutagenesis revealed that the full-length dimeric form and the metal ion Mg2+ are indispensable for the catalytic activity of Mtb-MazG. Thus, our work provides new insights into understanding the molecular basis of Mtb-MazG.

Genome-scale CRISPR-Cas9 knockout screening in nasopharyngeal carcinoma for radiosensitive and radioresistant genes.

BACKGROUND: Genome-scale CRISPR-Cas9 knockout screening may provide new insights into the mechanism underlying clinical radioresistance in nasopharyngeal carcinoma (NPC), which is remain largely unknown. Our objective was to screen the functional genes associated with radiosensitivity and radioresistance in NPC, laying a foundation for further research on its functional mechanismand. METHODS: CRISPR-Cas9 library lentivirus screening in radiation-treated NPC cells was combined with second-generation sequence technology to identify functional genes, which were further validated in radioresistant NPC cells and patient tissues. RESULTS: Eleven radiosensitive and radioresistant genes were screened. Among these genes, the expression of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower and TOMM20, CDKN2AIP, SNX22, and SP1 were higher in the radioresistant NPC cells (C666-1R, 5-8FR) (p < 0.05). CALD1 was highly expressed in C666-1R. Furthermore, we found knockout of FBLN5, FAM3C, MUS81 and DNAJC17 promoted the proliferation of NPC cells, while CDKN2AIP and SP1 had the opposed results (p < 0.05). This result was verified in NPC patient tissues. Meanwhile, KEGG analysis showed that the Fanconi anemia pathway and the TGF-ß signaling pathway possibly contributed to radiosensitivity or radioresistance in NPC. CONCLUSIONS: Nine genes involved in the radiosensitivity or radioresistance of NPC: four genes for radiosensitivity (FBLN5, FAM3C, MUS81, and DNAJC17), two genes for radioresistance (CDKN2AIP, SP1), two potential radioresistant genes (TOMM20, SNX22), and a potential radiosensitive gene (CALD1). Genome-scale CRISPR-Cas9 knockout screening for radiosensitive and radioresistant genes in NPC may provide new insights into the mechanisms underlying clinical radioresistance to improve the efficacy of radiotherapy for NPC.

Autoreactivity and broad neutralization of antibodies against HIV-1 are governed by distinct mutations: Implications for vaccine design strategies.

Many of the best HIV-1 broadly neutralizing antibodies (bnAbs) known have poly-/autoreactive features that disfavor normal B cell development and maturation, posing a major hurdle in developing an effective HIV-1 vaccine. Key to resolving this problem is to understand if, and to what extent, neutralization breadth-conferring mutations acquired by bnAbs contribute to their autoreactivity. Here, we back-mutated all known changes made by a prototype CD4 binding site-directed bnAb lineage, CH103-106, during its later maturation steps. Strikingly, of 29 mutations examined, only four were crucial for increased autoreactivity, with minimal or no impact on neutralization. Furthermore, three of these residues were clustered in the heavy chain complementarity-determining region 2 (HCDR2). Our results demonstrate that broad neutralization activity and autoreactivity in the CH103-106 bnAb lineage can be governed by a few, distinct mutations during maturation. This provides strong rationale for developing immunogens that favor bnAb lineages bearing "neutralization-only" mutations into current HIV-1 vaccine designs.

Structural insights of the elongation factor EF-Tu complexes in protein translation of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second-deadliest infectious disease worldwide. Emerging evidence shows that the elongation factor EF-Tu could be an excellent target for treating Mtb infection. Here, we report the crystal structures of Mtb EF-Tu•EF-Ts and EF-Tu•GDP complexes, showing the molecular basis of EF-Tu's representative recycling and inactive forms in protein translation. Mtb EF-Tu binds with EF-Ts at a 1:1 ratio in solution and crystal packing. Mutation and SAXS analysis show that EF-Ts residues Arg13, Asn82, and His149 are indispensable for the EF-Tu/EF-Ts complex formation. The GDP binding pocket of EF-Tu dramatically changes conformations upon binding with EF-Ts, sharing a similar GDP-exchange mechanism in E. coli and T. ther. Also, the FDA-approved drug Osimertinib inhibits the growth of M. smegmatis, H37Ra, and M. bovis BCG strains by directly binding with EF-Tu. Thus, our work reveals the structural basis of Mtb EF-Tu in polypeptide synthesis and may provide a promising candidate for TB treatment.

Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states.

RNA helicase DDX21 plays vital roles in ribosomal RNA processing and the regulation of host innate immunity during virus infection. Here, we describe the optimized protocols for nucleic acid-free protein purification and crystallization of DDX21 in its different unwinding states. Rational design of the flexible region within the helicase core, and biophysical approach to characterize interactions between DDX21 and RNA, leads to successful crystallization of DDX21. This protocol can be applied to the crystallography of other DExD/H-box RNA helicases. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Systematic Analysis of Chemokines Reveals CCL18 is a Prognostic Biomarker in Glioblastoma.

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor in adults, in which chemokines are often upregulated and may play pivotal roles in their development and progression. Chemokines are a large subfamily of cytokines with leukocyte chemotactic activities involved in various tumor progression. However, gene expression patterns of the chemokines on a global scale were not known in GBM. Methods: Differentially expressed chemokine genes in glioma and normal samples were screened by using The Cancer Genome Atlas (TCGA) database. Cox regression identified the prognosis-related genes in each glioma subtype. The protein expression levels of chemokines in 72 glioma tissues were detected by ELISA. Results: We found that the transcripts of seven chemokines, including CCL2, CCL8, CCL18, CCL28, CXCL1, CXCL5, and CXCL13, were highly expressed in GBM that evidenced by involving immune cell infiltration regulation and accompanied with worse outcomes of GBM patients. The prognostic nomogram construction demonstrated that CCL18 held the highest risk score in patients with GBM. Furthermore, experiments on 72 glioma tissue samples confirmed that CCL18 protein expression was positively associated with tumor grade and IDH1 status but inversely with glioma patients' overall survival (OS). Conclusion: Our study reveals comprehensive and comparable roles of chemokine members in glioblastoma, and identified CCL18 as a critical driver of GBM malignant behaviors, therefore providing a potential target for developing prognosis and therapy in human glioblastoma.

Immune Response on Optimal Timing and Fractionation Dose for Hypofractionated Radiotherapy in Non-Small-Cell Lung Cancer.

Background: The intervention timing of immune checkpoint inhibitors (ICIs) and radiotherapy fractionations are critical factors in clinical efficacy. This study aims to explore dynamic changes of the tumor immune microenvironment (TIME) after hypofractionated radiotherapy (HFRT) at different timepoints and fractionation doses in non-small-cell lung cancer (NSCLC). Methods: In the implanted mouse model, the experimental groups received HFRT 3.7 Gy × 4 F, 4.6 Gy × 3 F, 6.2 Gy × 2 F, and 10 Gy × 1 F, respectively, with the same biological equivalent dose (BED) of 20Gy. Tumor volume and survival time were compared with those of the control group. Flow cytometry was performed to detect immune cells and their PD-1/PD-L1 expressions using tail-tip blood at different timepoints and tumor tissues at 48 h after radiotherapy. In NSCLC patients, immune cells, PD-1/PD-L1, and cytokines were detected in peripheral blood for 4 consecutive days after different fractionation radiotherapy with the same BED of 40Gy. Results: Tumor volumes were significantly reduced in all experimental groups compared with the control group, and the survival time in 6.2 Gy × 2 F (p < 0.05) was significantly prolonged. In tail-tip blood of mice, CD8+ T counts increased from 48 h to 3 weeks in 4.6 Gy × 3 F and 6.2 Gy × 2 F, and CD8+ PD-1 shortly increased from 48 h to 2 weeks in 6.2 Gy × 2 F and 10 Gy × 1 F (p < 0.05). Dentritic cells (DCs) were recruited from 2 to 3 weeks (p < 0.01). As for NSCLC patients, CD8+ T counts and PD-1 expression increased from 24 h in 6.2 Gy × 4 F, and CD8+ T counts increased at 96 h in 10 Gy × 2 F (p < 0.05) in peripheral blood. DC cells were tentatively recruited at 48 h and enhanced PD-L1 expression from 24 h in both 6.2 Gy × 4 F and 10 Gy × 2 F (p < 0.05). Besides, serum IL-10 increased from 24 h in 6.2 Gy × 4 F (p < 0.05). Conversely, serum IL-4 decreased at 24 and 96 h in 10 Gy × 2 F (p < 0.05). Conclusion: HFRT induces the increase in CD8+ T cells and positive immune cytokine response in specific periods and fractionation doses. It was the optimal time window from 48 h to 2 weeks for the immune response, especially in 6.2 Gy fractionation. The best immune response was 96 h later in 10 Gy fractionation, delivering twice instead of a single dose. During this time window, the intervention of immunotherapy may achieve a better effect.