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Frontiers in Plant Science 2024
PubMed: 38938630
DOI: 10.3389/fpls.2024.1439668 -
Journal of Experimental & Clinical... Jun 2024Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are...
BACKGROUND
Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.
METHODS
We investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan-Meier plotter database and clinical breast cancer tissue samples.
RESULTS
We demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.
CONCLUSIONS
Our findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.
Topics: Humans; Triple Negative Breast Neoplasms; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Mice; Animals; Organelle Biogenesis; Phosphorylation; Glycogen Synthase Kinase 3 beta; Naphthoquinones; Female; Cancer-Associated Fibroblasts; Cell Line, Tumor; Mice, SCID; Neoplasm Metastasis; Mice, Inbred NOD; Mitochondria; Xenograft Model Antitumor Assays
PubMed: 38937832
DOI: 10.1186/s13046-024-03101-z -
Journal of Microbiology and... Jun 2024Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity... (Review)
Review
Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, polycystic kidney disease-like domain, collagen-binding domain, proprotein convertase domain, and β-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, increasing the degradation efficiency of collagen. Thermostable bacterial collagenolytic proteases function at high temperatures, which has the advantage of increasing the degradation efficiency because heat-denatured collagen is more susceptible to proteolysis and can minimize the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60°C. In this review, we present and summarize current research on the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms as well as the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of thermostable collagenolytic proteases in each family and comprehensively discuss the thermostable collagenolytic protease TSS.
PubMed: 38934777
DOI: 10.4014/jmb.2404.04051 -
ACS Chemical Biology Jun 2024TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and...
TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.
PubMed: 38934237
DOI: 10.1021/acschembio.4c00301 -
Cardiology Jun 2024High-molecular-weight (HMW) von Willebrand Factor (VWF) multimer deficiency occurs in classical low-flow, low-gradient (LF/LG) aortic stenosis (AS) due to shear...
INTRODUCTION
High-molecular-weight (HMW) von Willebrand Factor (VWF) multimer deficiency occurs in classical low-flow, low-gradient (LF/LG) aortic stenosis (AS) due to shear force-induced proteolysis. The prognostic value of HMW VWF multimer deficiency is unknown. Therefore, we sought to evaluate the impact of HMW VWF multimer deficiency on clinical outcome.
METHODS
In this prospective research study, a total of 83 patients with classical LF/LG AS were included. All patients underwent dobutamine-stress-echocardiography to distinguish true-severe (TS) from pseudo-severe (PS) classical LF/LG AS. HMW VWF multimer ratio was calculated using densitometric Western blot band quantification. The primary endpoint was all-cause mortality.
RESULTS
Mean age was 79 ± 9 years and TS classical LF/LG AS was diagnosed in 73% (n=61) and PS classical LF/LG AS in 27% (n=22) of all patients. Forty-six patients underwent aortic valve replacement (AVR) and 37 were treated conservatively. During a mean follow-up of 27 ± 17 months, 47 deaths occurred. Major bleeding complications after AVR (10/46; 22%) were more common in patients with HMW VWF multimer ratio <1 (8/17; 47%) in comparison to patients with a normal multimer pattern (2/29; 7%) at baseline (p=0.003). In a multivariable Cox regression analysis HMW VWF multimer deficiency was a predictor of all-cause mortality (HR: 3.02 [95% CI: 1.31-6.96], p=0.009) in the entire cohort. This association was driven by higher mortality rates in the AVR group (multivariable-adjusted HR: 9.4; 95%CI 2.0-43.4, p=0.004).
CONCLUSIONS
This is the first study to demonstrate the predictive value of HMW VWF multimer ratio for risk stratification in patients with classical LF/LG AS. HMW VWF multimer deficiency was associated with an increased risk of all-cause mortality and major bleeding complications after AVR.
PubMed: 38934149
DOI: 10.1159/000539731 -
Frontiers in Endocrinology 2024Tryptophan's (Trp) metabolites are undervalued markers of human health. Their serum concentrations are modified by physical exercise and other factors, among which...
INTRODUCTION
Tryptophan's (Trp) metabolites are undervalued markers of human health. Their serum concentrations are modified by physical exercise and other factors, among which fasting has a well-documented role. Although this mechanism is hardly explored, thus, the study aimed to determine the effect of the 8-day fasting period and the impact of such a procedure on a single bout of an endurance exercise on the concentration of kynurenine pathway (KP) metabolites.
METHODS
10 participants fasted for 8 days, and 10 as a control group participated in the study. The exercise was performed at baseline after an overnight fast and repeated post 8 days.
RESULTS
The 8 days of fasting increased the resting 3-hydroxy-L-kynurenine (3HK), picolinic acid (PA), kynurenic acid (KYNA), and xanthurenic acid (XA) serum concentration. Also elevated phenylalanine (Phe) and tyrosine (Tyr) levels were recorded, suggesting expanded proteolysis of muscle proteins. In turn, physical activity caused a decrease in the concentration of 3-hydroxyanthranilic acid (3HAA) and PA after fasting. The obtained results were not recorded in controls.
CONCLUSION
The results of this study show that the health-promoting effects of fasting are associated with changes in the KYN pathway. The increase in the concentration of PA and XA metabolites following fasting is capable of penetrating the blood-brain barrier, and KYNA, which initiates several beneficial changes, supports this assumption.
Topics: Humans; Male; Fasting; Kynurenine; Exercise; Adult; Young Adult; Rest; Healthy Volunteers; Kynurenic Acid; Tryptophan; Biomarkers; Picolinic Acids
PubMed: 38933822
DOI: 10.3389/fendo.2024.1403491 -
Nucleic Acids Research Jun 2024The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ...
The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.
PubMed: 38932681
DOI: 10.1093/nar/gkae539 -
Plants (Basel, Switzerland) Jun 2024Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic...
Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.
PubMed: 38931128
DOI: 10.3390/plants13121696 -
Molecules (Basel, Switzerland) Jun 2024Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For...
Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.
Topics: Humans; Cyclophilin A; Cyclosporine; Proteolysis; T-Lymphocytes; Lymphocyte Activation; HeLa Cells; Cell Proliferation; Immunosuppressive Agents; Proteolysis Targeting Chimera
PubMed: 38930843
DOI: 10.3390/molecules29122779 -
Microorganisms Jun 2024Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug...
Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug toxicity, is essential to eradicating HIV. This study investigated the potential of fractionated crude extracts of as immunomodulatory or anti-HIV drug candidates. Solid-phase extraction (SPE) was used to fractionate PO4PR2 using three different columns: MAX (Mixed-mode, strong Anion-eXchange), MCX (Mixed-mode, strong Cation-eXchange), and HLB (Hydrophilic-Lipophilic Balance) with methanol gradient methods (5%, 45%, and 95%). An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to assess the cell viability and cytotoxicity of the fractionated crude extract PO4PR2 in the TZM-bl cell lines. This was followed by a luciferase-based antiviral assay to assess the antiviral activity of PO4PR2. A time of addition (TOA) assay was performed to ascertain the mechanism of inhibition employed by the fractionated crude extract of PO4PR2 in the HIV life cycle. The p24 titer was determined using an ELISA, while a luciferase-based antiviral assay was used to evaluate the HIV percentage inhibition for different HIV-1 replication cycles. The TOA assay was established using antiviral drugs that target different sites in the HIV replication cycle. These included maraviroc, azidothymidine, raltegravir, and amprenavir. The immunomodulatory effect of the fractionated crude extracts on CD4+ T cells was measured by a flow cytometric analysis, for which fluorochrome-labelled monoclonal antibodies were used as markers for activation (CD38 and HLA-DR) and exhaustion (PD-1). The MCX fraction demonstrated a more significant anti-HIV inhibition than that of the fractions generated in other columns, with an IC of 0.3619 µg/mL, an HIV inhibition of 77%, 5% HLB (IC: 0.7232 µg/mL; HIV inhibition of 64%), and 5% MAX (IC: 5.240 µg/mL; HIV inhibition of 67%). It was evident from the time of addition data that the crude extract and the 5% MCX fraction inhibited viral binding (68%), reverse transcription (75%), integration (98%), and proteolysis (77%). It was shown that (the MCX fraction) have a significant inhibitory effect on reverse transcription (75% HIV inhibition) and integration (100% HIV inhibition). The 5% MCX ( = 0.0062), 5% HLB ( = 0.0269), and 5% MAX ( = 0.0117) fractionated crude extracts had low levels of CD4+ T cell (CD38 + HLA-DR+) activation compared to those of the AZT treatment, while CD4+ T cell activation was insignificant. The 5% MAX and HLB fractions may possess immunomodulatory compounds with less anti-HIV-1 activity. could be a key source of innovative anti-HIV drugs with immunomodulatory characteristics.
PubMed: 38930532
DOI: 10.3390/microorganisms12061150