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Scientific Reports Jun 2024The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen...
The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.
Topics: Hypoxia-Inducible Factor 1, alpha Subunit; Animals; Cathepsin L; Proteolysis; Rabbits; Oxygen; Humans; Reticulocytes; Basic Helix-Loop-Helix Transcription Factors; Hydroxylation
PubMed: 38926538
DOI: 10.1038/s41598-024-65537-9 -
Nature Communications Jun 2024METTL3 is the catalytic subunit of the methyltransferase complex, which mediates mA modification to regulate gene expression. In addition, METTL3 regulates transcription...
METTL3 is the catalytic subunit of the methyltransferase complex, which mediates mA modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of the methyltransferase complex are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated mA sites. In summary, our results report a coordination of mA-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming.
Topics: Methyltransferases; Chromatin; Cellular Senescence; Humans; Stress Granules; Hexokinase; RNA, Messenger; Adenosine; HEK293 Cells; Metabolic Reprogramming; Phase Separation
PubMed: 38926365
DOI: 10.1038/s41467-024-49745-5 -
The Journal of Biological Chemistry Jun 2024Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide...
Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor (GPCR) complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss-of-function or constitutive gain-of-function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed clinically-relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics (MD) simulations, which observed changes in interaction networks between GαS switch regions. Further, saturation-transfer difference NMR (STD-NMR) spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.
PubMed: 38925329
DOI: 10.1016/j.jbc.2024.107497 -
The Journal of Biological Chemistry Jun 2024Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for discovery of novel drugs to tackle ART resistant parasites. Concentrated efforts...
Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for discovery of novel drugs to tackle ART resistant parasites. Concentrated efforts towards ART resistance mechanism indicated a strong molecular link of ART resistance with up-regulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6), and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA approved drug 'Biperiden' that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART sensitive Pf3D7 and resistant Pf3D7k13 strains. Ring survival assays that are clinically relevant to analyse ART resistance in Pf3D7k13 parasites demonstrate the potency of BPD to inhibit growth of survivor parasites. Overall, our study provides first evidence towards the role of PfPFDs in ART resistance mechanism, and opens new avenues for the management of resistant parasite.
PubMed: 38925325
DOI: 10.1016/j.jbc.2024.107496 -
PloS One 2024The development of a cancer vaccine has become an essential focus in the field of medical biotechnology and immunology. In our study, the NY-SAR-35 cancer/testis antigen...
Bioinformatics design of a peptide vaccine containing sarcoma antigen NY-SAR-35 epitopes against breast cancer and evaluation of its immunological function in BALB/c mouse model.
The development of a cancer vaccine has become an essential focus in the field of medical biotechnology and immunology. In our study, the NY-SAR-35 cancer/testis antigen was targeted to design a novel peptide vaccine using bioinformatics tools, and BALB/c mice were used to evaluate the vaccine's immunological function. This evaluation involved assessing peptide-specific IgG levels in the serum via ELISA and measuring the levels of IFN-γ, IL-4, and granzyme B in the supernatant of cultured splenocytes. The final vaccine construct consisted of two T lymphocyte epitopes linked by the AAY linker. This construct displayed high antigenicity, non-allergenicity, non-toxicity, stability, and ability to induce IFN-γ and IL-4. It showed stable dynamics with both human MHC-I and II molecules, as well as mouse MHC-II molecules, and revealed strong Van der Waals and electrostatic energies. Emulsifying our peptide vaccine in incomplete Freund's adjuvant resulted in a remarkable increase in the levels of IgG. The splenocytes of mice that received the combination of peptide and adjuvant displayed a noteworthy increase in IFN-γ, IL-4, and granzyme B secretion. Additionally, their lymphocytes exhibited higher proliferation rates compared to the control group. Our data demonstrated that our vaccine could stimulate a robust immune response, making it a promising candidate for cancer prevention. However, clinical trials are necessary to assess its efficacy in humans.
Topics: Animals; Cancer Vaccines; Mice, Inbred BALB C; Mice; Female; Antigens, Neoplasm; Computational Biology; Humans; Vaccines, Subunit; Breast Neoplasms; Epitopes, T-Lymphocyte; Interleukin-4; Interferon-gamma; Immunoglobulin G; Granzymes; Disease Models, Animal; Protein Subunit Vaccines
PubMed: 38923980
DOI: 10.1371/journal.pone.0306117 -
ELife Jun 2024During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid...
During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.
Topics: Dendritic Cells; Glycolysis; Monocytes; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mycobacterium tuberculosis; Cell Movement; Animals; Tuberculosis; Mice; Toll-Like Receptor 2; Mice, Inbred C57BL; Female
PubMed: 38922679
DOI: 10.7554/eLife.89319 -
MBio Jun 2024The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." growth...
UNLABELLED
The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." growth is inhibited by calprotectin , and calprotectin is localized to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in , which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of to the presence of calprotectin. ClpP contributes to pathogenesis in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for to outcompete the host for metals.
IMPORTANCE
is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of to survive in a metal-limited environment.
PubMed: 38920392
DOI: 10.1128/mbio.01389-24 -
Frontiers in Cellular and Infection... 2024The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a highly contagious respiratory disease...
The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a highly contagious respiratory disease with widespread societal impact. The symptoms range from cough, fever, and pneumonia to complications affecting various organs, including the heart, kidneys, and nervous system. Despite various ongoing efforts, no effective drug has been developed to stop the spread of the virus. Although various types of medications used to treat bacterial and viral diseases have previously been employed to treat COVID-19 patients, their side effects have also been observed. The way SARS-CoV-2 infects the human body is very specific, as its spike protein plays an important role. The S subunit of virus spike protein cleaved by human proteases, such as furin protein, is an initial and important step for its internalization into a human host. Keeping this context, we attempted to inhibit the furin using phytochemicals that could produce minimal side effects. For this, we screened 408 natural phytochemicals from various plants having antiviral properties, against furin protein, and molecular docking and dynamics simulations were performed. Based on the binding score, the top three compounds (robustaflavone, withanolide, and amentoflavone) were selected for further validation. MM/GBSA energy calculations revealed that withanolide has the lowest binding energy of -57.2 kcal/mol followed by robustaflavone and amentoflavone with a binding energy of -45.2 kcal/mol and -39.68 kcal/mol, respectively. Additionally, ADME analysis showed drug-like properties for these three lead compounds. Hence, these natural compounds robustaflavone, withanolide, and amentoflavone, may have therapeutic potential for the management of SARS-CoV-2 by targeting furin.
Topics: Furin; Phytochemicals; Humans; Molecular Docking Simulation; SARS-CoV-2; Antiviral Agents; COVID-19 Drug Treatment; Protease Inhibitors; Spike Glycoprotein, Coronavirus; COVID-19; Protein Binding
PubMed: 38919703
DOI: 10.3389/fcimb.2024.1391288 -
Frontiers in Immunology 2024Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several...
BACKGROUND
Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several chronic inflammatory diseases. However, increased liver fibrosis has been reported in mice lacking IL-15 receptor alpha chain (IL-15Rα), suggesting an anti-fibrogenic role for IL-15. As myeloid cells are key players in liver fibrosis and IL-15 signaling can occur independently of IL-15Rα, we investigated the requirement of IL-15 and IL-15Rα in liver fibrosis.
METHODS
We induced liver fibrosis in , and wildtype C57BL/6 mice by the administration of carbon tetrachloride (CCl). Liver fibrosis was evaluated by Sirius red and Mason's trichrome staining and α-smooth muscle acting immunostaining of myofibroblasts. Gene expression of collagens, matrix modifying enzymes, cytokines and chemokines was quantified by RT-qPCR. The phenotype and the numbers of intrahepatic lymphoid and myeloid cell subsets were evaluated by flow cytometry.
RESULTS
Both and mice developed markedly reduced liver fibrosis compared to wildtype control mice, as revealed by reduced collagen deposition and myofibroblast content. mice showed further reduction in collagen deposition compared to mice. However, and genes were similarly induced in the fibrotic livers of wildtype, and mice, although notable variations were observed in the expression of matrix remodeling enzymes and chemokines. As expected, and mice showed markedly reduced numbers of NK cells compared to wildtype mice. They also showed markedly less staining of CD45 immune cells and CD68 macrophages, and significantly reduced inflammatory cell infiltration into the liver, with fewer pro-inflammatory and anti-inflammatory monocyte subsets compared to wildtype mice.
CONCLUSION
Our findings indicate that IL-15 exerts its profibrogenic role in the liver by promoting macrophage activation and that this requires trans-presentation of IL-15 by IL-15Rα.
Topics: Animals; Interleukin-15; Mice; Carbon Tetrachloride; Interleukin-15 Receptor alpha Subunit; Liver Cirrhosis; Mice, Knockout; Mice, Inbred C57BL; Disease Models, Animal; Male; Liver; Cytokines; Receptors, Interleukin-15
PubMed: 38919611
DOI: 10.3389/fimmu.2024.1404891 -
BMB Reports Jun 2024The heterotrimeric molecular motor kinesin-2 is involved in the microtubule-dependent transport of intracellular cargo. It consists of two distinct motor subunits...
The heterotrimeric molecular motor kinesin-2 is involved in the microtubule-dependent transport of intracellular cargo. It consists of two distinct motor subunits (KIF3A, and KIF3B) and a non-motor subunit, kinesin-associated protein 3 (KAP3). The cargo-binding domain (CBD) at the carboxyl (C)-terminus of KIF3s plays an important role in the interaction with several different binding proteins. To identify the binding proteins for heterotrimeric kinesin-2, we performed a yeast two-hybrid screen and found a new interaction with Disables-1 (Dab1), the intracellular adaptor protein of reelin receptors. Dab1 bound to the CBD of KIF3A, but did not interact with the C-terminal domain of KIF3B, KIF5B, KIF17 or KAP3. The phosphotyrosine binding (PTB) domain-containing region of Dab1 is essential for the interaction with KIF3A. KIF3A interacted with GST-Dab1, and GST-CaMKIIα, but did not interact with GST-apolipoprotein E receptor 2 (ApoER2)-C or with GST alone. When co-expressed in HEK-293T cells, KIF3A co-precipitated with Dab1, but not with KIF5B. Dab1 and KIF3A were co-localized in cultured cells. We also identified deduced cell surface expression of ApoER2 in KIF3A dominant-negative cells. These results suggest that the KIF3A plays a role in the intracellular trafficking of ApoER2 to the cell surface.
PubMed: 38919020
DOI: No ID Found