Crystal structure of Bak bound to the BH3 domain of Bnip5, a noncanonical BH3 domain-containing protein.

The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.

De novo biosynthesis of 2'-fucosyllactose by bioengineered Corynebacterium glutamicum.

2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

BAP1 controls mesenchymal stem cell migration by inhibiting the ERK signaling pathway.

Mesenchymal stem cells (MSCs) have remarkable potential in regenerative medicine owing to their stem-like characteristics and immunosuppressive properties. Much effort has been devoted to enhancing the efficacy of MSC therapy by enhancing MSC migration. In this study, we identified deubiquitinase BRCA1-associated protein 1 (BAP1) as an inhibitor of MSC migration. Using deubiquitinase siRNA library screening based on an in vitro wound healing assay, we found that silencing BAP1 significantly augmented MSC migration. Conversely, BAP1 overexpression reduced the migration and invasion capabilities of MSCs. BAP1 depletion in MSCs upregulates ERK phosphorylation, thereby increasing the expression of the migration factor osteopontin. Further examination revealed that BAP1 interacts with phosphorylated ERK1/2, deubiquitinating their ubiquitins, and thus attenuating the ERK signaling pathway. Overall, our study highlights the critical role of BAP1 in regulating MSC migration through its deubiquitinase activity and suggests a novel approach to improve the therapeutic potential of MSCs in regenerative medicine.

The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodelling lipid metabolism.

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1.

Bak is a critical executor of apoptosis belonging to the Bcl-2 protein family. Bak contains a hydrophobic groove where the BH3 domain of proapoptotic Bcl-2 family members can be accommodated, which initiates its activation. Once activated, Bak undergoes a conformational change to oligomerize, which leads to mitochondrial destabilization and the release of cytochrome c into the cytosol and eventual apoptotic cell death. In this study, we investigated the molecular aspects and functional consequences of the interaction between Bak and peroxisomal testis-specific 1 (Pxt1), a noncanonical BH3-only protein exclusively expressed in the testis. Together with various biochemical approaches, this interaction was verified and analyzed at the atomic level by determining the crystal structure of the Bak-Pxt1 BH3 complex. In-depth biochemical and cellular analyses demonstrated that Pxt1 functions as a Bak-activating proapoptotic factor, and its BH3 domain, which mediates direct intermolecular interaction with Bak, plays a critical role in triggering apoptosis. Therefore, this study provides a molecular basis for the Pxt1-mediated novel pathway for the activation of apoptosis and expands our understanding of the cell death signaling coordinated by diverse BH3 domain-containing proteins.

Exploitation of a novel adjuvant for polymyxin B against multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Although polymyxin has been used as a last-resort antibiotic against resistant bacteria, its use is restricted due to nephrotoxicity and neurotoxicity. While the present antibiotic resistance issue compels clinicians to reconsider polymyxin use in severe illness cases, polymyxin-resistant microorganisms exert an effect. OBJECTIVES: To address the issue of antibiotic resistance, the cycle of developing new antibiotics to counteract emerging resistance must be discontinued. Here we tried to develop novel therapies that do not rely on direct antimicrobial activity and thus do not promote antibiotic resistance. METHODS: By a high-throughout screening system based on bacterial respiration, chemical compounds accelerating the antimicrobial effects of polymyxin B were screened. In vitro and in vivo tests were performed to validate adjuvanticity. In addition, membrane depolarization and total transcriptome analysis were used to determine molecular mechanisms. RESULTS: PA108, a newly discovered chemical compound, was used to eradicate polymyxin-resistant A. baumannii and three other species in the presence of polymyxin B at concentrations less than the MIC. Since this molecule lacks self-bactericidal action, we hypothesized that PA108 acts as an antibiotic adjuvant, enhancing the antimicrobial activity of polymyxin B against resistant bacteria. At working concentrations, no toxicity was observed in cell lines or mice, although co-treatment with PA108 and polymyxin B increased survival of infected mouse and decreased bacterial loads in organs. CONCLUSIONS: Boosting antibiotic efficiency through the use of antibiotic adjuvants holds significant promise for tackling the rise in bacterial antibiotic resistance.

Antioxidants prevent particulate matter-induced senescence of lung fibroblasts.

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

Structural and biochemical analyses of Bcl-xL in complex with the BH3 domain of peroxisomal testis-specific 1.

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

Mannose-6-phosphate glycan for lysosomal targeting: various applications from enzyme replacement therapy to lysosome-targeting chimeras.

A lysosome, an acidic membrane-bound organelle, contains hydrolytic enzymes to digest macromolecules for recycling. Many lysosomal enzymes (LEs) traffic to the lysosome through the mannose-6-phosphate (M6P)-dependent pathway. Some mannose residues of high-mannose type N-glycans on LEs can be phosphorylated in the Golgi apparatus through two-step enzyme reactions. The consequent M6P moiety is recognized by M6P receptors (MPRs) on the trans-Golgi network membrane and delivered through the endo-lysosomal pathway. On the other hand, secreted LEs containing M6P glycans can be recaptured by MPRs on the plasma membrane and targeted to the lysosome. Enzyme replacement therapy (ERT) for lysosomal storage diseases exploits this M6P-MPR-dependent endocytosis to deliver recombinant enzymes to lysosomes. This review discusses various engineering and application technologies using M6P's lysosomal targeting. Glyco-engineering for increasing M6P contents developed 'Bio-better' ERT enzymes with enhanced therapeutic efficacy. M6P-decorated peptides, proteins, liposomes, and nanoparticles have been developed for drug delivery and subcellular imaging. A recently developed lysosome-targeting chimera uses an M6P-based bifunctional binder to degrade specific extracellular and membrane proteins. The success and efficiency of M6P-based lysosomal targeting will boost further technological developments with new applications in the biomedical field.

Prebiotic activities of dextran from Leuconostoc mesenteroides SPCL742 analyzed in the aspect of the human gut microbial ecosystem.

The aim of this study was to investigate the prebiotic activities of dextran (LM742) produced by Leuconostoc mesenteroides SPCL742 in the aspect of the human gut microbial ecosystem focusing on microbiome and metabolome changes in in vitro colonic fermentation. LM742 dextran had a medium-chain structure with the molecular weight of 1394.87 kDa (DP = 7759.22) and α-1,6 and α-1,3 linkages with a 26.11 : 1 ratio. The LM742 dextran was resistent to digestive enzymes in the human gastrointestinal conditions. The individual cultivation of 30 intestinal bacteria with LM742 dextran showed the growth of Bacteroides spp., whereas in vitro human fecal fermentation with LM742 exhibited the symbiotic growth of Bacteroides spp. and beneficial bacteria such as Bifidobacterium spp. Further co-cultivation of Bacteroides xylanisolvens and several probiotics indicated that B. xylanisolvens provides a cross-feeding of dextran to probiotics. In fecal fermentation, LM742 dextran resulted in increased concentrations of short-chain fatty acids, valerate and pantothenate, but it rarely affected the conversion of betaine to trimethylamine. Lastly, LM742 dextran inhibited the adhesion of pathogenic E. coli to human epithelial cells. Taken together, these results demonstrate the prebiotic potential of LM742 dextran as a health-beneficial polysaccharide in the human intestine.

Necroptosis molecular mechanisms: Recent findings regarding novel necroptosis regulators.

Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein-protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis.

Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer.

Ferroptosis is an iron-dependent regulated necrosis mediated by lipid peroxidation. Cancer cells survive under metabolic stress conditions by altering lipid metabolism, which may alter their sensitivity to ferroptosis. However, the association between lipid metabolism and ferroptosis is not completely understood. In this study, we found that the expression of elongation of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1) is up-regulated in mesenchymal-type gastric cancer cells (GCs), leading to ferroptosis sensitization. In contrast, these enzymes are silenced by DNA methylation in intestinal-type GCs, rendering cells resistant to ferroptosis. Lipid profiling and isotope tracing analyses revealed that intestinal-type GCs are unable to generate arachidonic acid (AA) and adrenic acid (AdA) from linoleic acid. AA supplementation of intestinal-type GCs restores their sensitivity to ferroptosis. Based on these data, the polyunsaturated fatty acid (PUFA) biosynthesis pathway plays an essential role in ferroptosis; thus, this pathway potentially represents a marker for predicting the efficacy of ferroptosis-mediated cancer therapy.

Whole-Genome Sequence of Lactobacillus plantarum SPC-SNU 72-2 as a Probiotic Starter for Sourdough Fermentation.

We report the whole-genome sequence of Lactobacillus plantarum SPC-SNU 72-2, a probiotic starter for sourdough. Genome sequencing was completed using the Pacific Biosciences RS II and Illumina platforms. This study will facilitate the understanding of microbial characteristics of L. plantarum SPC-SNU 72-2 and its roles during sourdough fermentation.

Evaluation of 2,3-Butanediol Production from Red Seaweed Gelidium amansii Hydrolysates Using Engineered Saccharomyces cerevisiae.

Hyper-thermal (HT) acid hydrolysis of red seaweed Gelidium amansii was performed using 12% (w/v) slurry and an acid mix concentration of 180 mM at 150°C for 10 min. Enzymatic saccharification when using a combination of Celluclast 1.5 L and CTec2 at a dose of 16 U/ml led to the production of 12.0 g/l of reducing sugar with an efficiency of enzymatic saccharification of 13.2%. After the enzymatic saccharification, 2,3-butanediol (2,3-BD) fermentation was carried out using an engineered S. cerevisiae strain. The use of HT acid-hydrolyzed medium with 1.9 g/l of 5-hydroxymethylfurfural showed a reduction in the lag time from 48 to 24 h. The 2,3-BD concentration and yield coefficient at 72 h were 14.8 g/l and 0.30, respectively. Therefore, HT acid hydrolysis and the use of the engineered S. cerevisiae strain can enhance the overall 2,3-BD yields from G. amansii seaweed.

Identification of MYC as an antinecroptotic protein that stifles RIPK1-RIPK3 complex formation.

The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1-RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

Post-Translational Regulation of ARF: Perspective in Cancer.

Tumorigenesis can be induced by various stresses that cause aberrant DNA mutations and unhindered cell proliferation. Under such conditions, normal cells autonomously induce defense mechanisms, thereby stimulating tumor suppressor activation. ARF, encoded by the CDKN2a locus, is one of the most frequently mutated or deleted tumor suppressors in human cancer. The safeguard roles of ARF in tumorigenesis are mainly mediated via the MDM2-p53 axis, which plays a prominent role in tumor suppression. Under normal conditions, low p53 expression is stringently regulated by its target gene, MDM2 E3 ligase, which induces p53 degradation in a ubiquitin-proteasome-dependent manner. Oncogenic signals induced by MYC, RAS, and E2Fs trap MDM2 in the inhibited state by inducing ARF expression as a safeguard measure, thereby activating the tumor-suppressive function of p53. In addition to the MDM2-p53 axis, ARF can also interact with diverse proteins and regulate various cellular functions, such as cellular senescence, apoptosis, and anoikis, in a p53-independent manner. As the evidence indicating ARF as a key tumor suppressor has been accumulated, there is growing evidence that ARF is sophisticatedly fine-tuned by the diverse factors through transcriptional and post-translational regulatory mechanisms. In this review, we mainly focused on how cancer cells employ transcriptional and post-translational regulatory mechanisms to manipulate ARF activities to circumvent the tumor-suppressive function of ARF. We further discussed the clinical implications of ARF in human cancer.

Antibody-secreting macrophages generated using CpG-free plasmid eliminate tumor cells through antibody-dependent cellular phagocytosis.

The non-viral delivery of genes into macrophages, known as hard-to-transfect cells, is a challenge. In this study, the microporation of a CpG-free and small plasmid (pCGfd-GFP) showed high transfection efficiency, sustainable transgene expression, and good cell viability in the transfections of Raw 264.7 and primary bone marrow-derived macrophages. The non-viral method using the pCGfd vector encoding anti-EGFR single-chain Fv fused with Fc (scFv-Fc) generated the macrophages secreting anti-EGFR scFv-Fc. These macrophages effectively phagocytized tumor cells expressing EGFR through the antibody-dependent mechanism, as was proved by experiments using EGFR-knockout tumor cells. Finally, peri-tumoral injections of anti-EGFR scFv-Fc-secreting macrophages were shown to inhibit tumor growth in the xenograft mouse model. [BMB Reports 2020; 53(8): 442-447].

Beclin 1 functions as a negative modulator of MLKL oligomerisation by integrating into the necrosome complex.

Necroptosis is a form of regulated cell death caused by formation of the necrosome complex. However, the factors modulating this process and the systemic pathophysiological effects of necroptosis are yet to be understood. Here, we identified that Beclin 1 functions as an anti-necroptosis factor by being recruited into the necrosome complex upon treatment with TNFα, Smac mimetic, and pan-caspase inhibitor and by repressing MLKL oligomerisation, thus preventing the disruption of the plasma membrane. Cells ablated or knocked-out for Beclin 1 become sensitised to necroptosis in an autophagy-independent manner without affecting the necrosome formation itself. Interestingly, the recruitment of Beclin 1 into the necrosome complex is dependent on the activation and phosphorylation of MLKL. Biochemically, the coiled-coil domain (CCD) of Beclin 1 binds to the CCD of MLKL, which restrains the oligomerisation of phosphorylated MLKL. Finally, Beclin 1 depletion was found to promote necroptosis in leukaemia cells and enhance regression of xenografted-tumour upon treatment with Smac mimetics and caspase inhibitors. These results suggest that Beclin 1 functions as a negative regulator in the execution of necroptosis by suppressing MLKL oligomerisation.

Pericardial Recess: Computed Tomography Findings of Varying Disorders.

A pericardial recess is frequently seen in patients undergoing chest computed tomography (CT). It is important to be aware of the normal anatomy of the pericardium as it is often mistaken for normal variants and/or disease. Therefore, we will describe the anatomy and location of the pericardial recess and the specific findings in various diseases associated with the pericardial recess.