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The Journal of Biological Chemistry May 2024The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel...
The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell-types. NDRG1 overexpression decreased β-catenin and down-regulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 that are classically induced by GSK-3β were significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression up-regulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. In fact, PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. In fact, the association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the down-regulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), that is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that up-regulate PKCα and inhibit WNT signaling.
PubMed: 38815861
DOI: 10.1016/j.jbc.2024.107417 -
Microbiology Spectrum May 2024is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates...
is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the lusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during infection.IMPORTANCE is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a -secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of infections but also in understanding the role of TRAF7 in cancer.
PubMed: 38814079
DOI: 10.1128/spectrum.00453-24 -
ELife May 2024Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important...
Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from -null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.
Topics: Animals; Female; Pregnancy; Genomic Imprinting; Mice; Polycomb Repressive Complex 2; Fetal Development; Placenta; Oocytes; Amino Acid Transport System A
PubMed: 38813868
DOI: 10.7554/eLife.81875 -
The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.Haematologica May 2024Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a...
Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, yet the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/plateletspecific knockout of PALLD in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain (DBD) and Src homology 2 (SH2) domain via Immunoglobulin domain 3 (Ig3). Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.
PubMed: 38813732
DOI: 10.3324/haematol.2024.285242 -
Disease Models & Mechanisms May 2024Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments (OS), which function as platforms for...
Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments (OS), which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lack OSs and undergo apoptosis. Interestingly, zebrafish mak mutants fail to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies are specified. These data suggest that MAK contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of MAK is critical in ciliary axoneme development and photoreceptor survival. Thus, MAK is required for ciliogenesis and OS formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.
PubMed: 38813692
DOI: 10.1242/dmm.050618 -
Cardiovascular Diabetology May 2024Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for...
BACKGROUND
Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for cardiovascular diseases. Although an association between ferroptosis and vascular calcification has been reported, the role and mechanism of iron overload in vascular calcification are still poorly understood. Specifically, further in-depth research is required on whether metalloproteins SLC39a14 and SLC39a8 are involved in ferroptosis induced by iron overload.
METHODS
R language was employed for the differential analysis of the dataset, revealing the correlation between ferroptosis and calcification. The experimental approaches encompassed both in vitro and in vivo studies, incorporating the use of iron chelators and models of iron overload. Additionally, gain- and loss-of-function experiments were conducted to investigate iron's effects on vascular calcification comprehensively. Electron microscopy, immunofluorescence, western blotting, and real-time polymerase chain reaction were used to elucidate how Slc39a14 and Slc39a8 mediate iron overload and promote calcification.
RESULTS
Ferroptosis was observed in conjunction with vascular calcification (VC); the association was consistently confirmed by in vitro and in vivo studies. Our results showed a positive correlation between iron overload in VSMCs and calcification. Iron chelators are effective in reversing VC and iron overload exacerbates this process. The expression levels of the metal transport proteins Slc39a14 and Slc39a8 were significantly upregulated during calcification; the inhibition of their expression alleviated VC. Conversely, Slc39a14 overexpression exacerbates calcification and promotes intracellular iron accumulation in VSMCs.
CONCLUSIONS
Our research demonstrates that iron overload occurs during VC, and that inhibition of Slc39a14 and Slc39a8 significantly relieves VC by intercepting iron overload-induced ferroptosis in VSMCs, providing new insights into the VC treatment.
Topics: Ferroptosis; Vascular Calcification; Animals; Cation Transport Proteins; Myocytes, Smooth Muscle; Disease Models, Animal; Muscle, Smooth, Vascular; Mice, Inbred C57BL; Iron Chelating Agents; Signal Transduction; Male; Humans; Iron; Iron Overload
PubMed: 38812011
DOI: 10.1186/s12933-024-02224-z -
Acta Neuropathologica Communications May 2024Neurons pose a particular challenge to degradative processes like autophagy due to their long and thin processes. Autophagic vesicles (AVs) are formed at the tip of the...
Neurons pose a particular challenge to degradative processes like autophagy due to their long and thin processes. Autophagic vesicles (AVs) are formed at the tip of the axon and transported back to the soma. This transport is essential since the final degradation of the vesicular content occurs only close to or in the soma. Here, we established an in vivo live-imaging model in the rat optic nerve using viral vector mediated LC3-labeling and two-photon-microscopy to analyze axonal transport of AVs. Under basal conditions in vivo, 50% of the AVs are moving with a majority of 85% being transported in the retrograde direction. Transport velocity is higher in the retrograde than in the anterograde direction. A crush lesion of the optic nerve results in a rapid breakdown of retrograde axonal transport while the anterograde transport stays intact over several hours. Close to the lesion site, the formation of AVs is upregulated within the first 6 h after crush, but the clearance of AVs and the levels of lysosomal markers in the adjacent axon are reduced. Expression of p150Glued, an adaptor protein of dynein, is significantly reduced after crush lesion. In vitro, fusion and colocalization of the lysosomal marker cathepsin D with AVs are reduced after axotomy. Taken together, we present here the first in vivo analysis of axonal AV transport in the mammalian CNS using live-imaging. We find that axotomy leads to severe defects of retrograde motility and a decreased clearance of AVs via the lysosomal system.
Topics: Animals; Axonal Transport; Optic Nerve; Rats; Autophagy; Optic Nerve Injuries; Male; Axons; Nerve Degeneration; Rats, Sprague-Dawley; Female
PubMed: 38812004
DOI: 10.1186/s40478-024-01791-2 -
Journal of Nanobiotechnology May 2024Advanced hepatocellular carcinoma (HCC) can be treated with sorafenib, which is the primary choice for targeted therapy. Nevertheless, the effectiveness of sorafenib is...
BACKGROUND
Advanced hepatocellular carcinoma (HCC) can be treated with sorafenib, which is the primary choice for targeted therapy. Nevertheless, the effectiveness of sorafenib is greatly restricted due to resistance. Research has shown that exosomes and circular RNAs play a vital role in the cancer's malignant advancement. However, the significance of exosomal circular RNAs in the development of resistance to sorafenib in HCC remains uncertain.
METHODS
Ultracentrifugation was utilized to isolate exosomes (Exo-SR) from the sorafenib-resistant HCC cells' culture medium. Transcriptome sequencing and differential expression gene analysis were used to identify the targets of Exo-SR action in HCC cells. To identify the targets of Exo-SR action in HCC cells, transcriptome sequencing and analysis of differential expression genes were employed. To evaluate the impact of exosomal circUPF2 on resistance to sorafenib in HCC, experiments involving gain-of-function and loss-of-function were conducted. RNA pull-down assays and mass spectrometry analysis were performed to identify the RNA-binding proteins interacting with circUPF2. RNA immunoprecipitation (RIP), RNA pull-down, electrophoretic mobility shift assay (EMSA), immunofluorescence (IF) -fluorescence in situ hybridization (FISH), and rescue assays were used to validate the interactions among circUPF2, IGF2BP2 and SLC7A11. Finally, a tumor xenograft assay was used to examine the biological functions and underlying mechanisms of Exo-SR and circUPF2 in vivo.
RESULTS
A novel exosomal circRNA, circUPF2, was identified and revealed to be significantly enriched in Exo-SR. Exosomes with enriched circUPF2 enhanced sorafenib resistance by promoting SLC7A11 expression and suppressing ferroptosis in HCC cells. Mechanistically, circUPF2 acts as a framework to enhance the creation of the circUPF2-IGF2BP2-SLC7A11 ternary complex contributing to the stabilization of SLC7A11 mRNA. Consequently, exosomal circUPF2 promotes SLC7A11 expression and enhances the function of system Xc- in HCC cells, leading to decreased sensitivity to ferroptosis and resistance to sorafenib.
CONCLUSIONS
The resistance to sorafenib in HCC is facilitated by the exosomal circUPF2, which promotes the formation of the circUPF2-IGF2BP2-SLC7A11 ternary complex and increases the stability of SLC7A11 mRNA. Focusing on exosomal circUPF2 could potentially be an innovative approach for HCC treatment.
Topics: Carcinoma, Hepatocellular; Humans; Exosomes; Liver Neoplasms; Sorafenib; RNA, Circular; Ferroptosis; Drug Resistance, Neoplasm; Cell Line, Tumor; Animals; Mice; RNA-Binding Proteins; Mice, Nude; Amino Acid Transport System y+; Antineoplastic Agents; Gene Expression Regulation, Neoplastic; Mice, Inbred BALB C
PubMed: 38811968
DOI: 10.1186/s12951-024-02582-6 -
Communications Biology May 2024Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as...
Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on polymerized microtubules deposited onto glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.
Topics: Kinesins; Animals; Rats; Neurites; Microtubules; Hippocampus; Cells, Cultured
PubMed: 38811803
DOI: 10.1038/s42003-024-06358-4 -
Communications Biology May 2024Two mammalian homologs of systemic RNA interference defective protein 1 (SID-1) (SIDT1/2) are suggested to function as double-stranded RNA (dsRNA) transporters for...
Two mammalian homologs of systemic RNA interference defective protein 1 (SID-1) (SIDT1/2) are suggested to function as double-stranded RNA (dsRNA) transporters for extracellular dsRNA uptake or for release of incorporated dsRNA from lysosome to cytoplasm. SIDT1/2 is also suggested to be involved in cholesterol transport and lipid metabolism. Here, we determine the cryo-electron microscopy structures of human SIDT1, homodimer in a side-by-side arrangement, with two distinct conformations, the cholesterol-bound form and the unbound form. Our structures reveal that the membrane-spanning region of SIDT1 harbors conserved histidine and aspartate residues coordinating to putative zinc ion, in a structurally similar manner to alkaline ceramidases or adiponectin receptors that require zinc for ceramidase activity. We identify that SIDT1 has a ceramidase activity that is attenuated by cholesterol binding. Observations from two structures suggest that cholesterol molecules serve as allosteric regulator that binds the transmembrane region of SIDT1 and induces the conformation change and the reorientation of the catalytic residues. This study represents a contribution to the elucidation of the cholesterol-mediated mechanisms of lipid hydrolytic activity and RNA transport in the SID-1 family proteins.
Topics: Humans; Cryoelectron Microscopy; Hydrolysis; Cholesterol; Lipid Metabolism; Protein Conformation; Models, Molecular; Protein Binding
PubMed: 38811802
DOI: 10.1038/s42003-024-06346-8