Cytoplasmic Protein Research Articles

Autophagy initiation triggers p150Glued–AP-2β interaction on the lysosomes and facilitates their transport

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued–AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued–AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued–AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued–AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued–AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

CLIP170 inhibits the metastasis and EMT of papillary thyroid cancer through the TGF-β pathway.

Metastasis poses a significant challenge in combating tumors. Even in papillary thyroid cancer (PTC), which typically exhibits a favorable prognosis, high recurrence rates are attributed to metastasis. Cytoplasmic linker protein 170 (CLIP170) functions as a classical microtubule plus-end tracking protein (+TIP) and has shown close association with cell migration. Nevertheless, the specific impact of CLIP170 on PTC cells remains to be elucidated. Our analysis of the GEO and TCGA databases unveiled an association between CLIP170 and the progression of PTC. To explore the impact of CLIP170 on PTC cells, we conducted various assays. We evaluated its effects through CCK-8, wound healing assay, and transwell assay after knocking down CLIP170. Additionally, the influence of CLIP170 on the cellular actin structure was examined via immunofluorescence; we further investigated the molecular expressions of epithelial-mesenchymal transition (EMT) and the transforming growth factor-β (TGF-β) signaling pathways through Western blotting and RT-qPCR. These findings were substantiated through an in vivo nude mouse model of lung metastasis. We observed a decreased expression of CLIP170 in PTC in contrast to normal thyroid tissue. Functionally, the knockdown of CLIP170 (CLIP170KD) notably enhanced the metastatic potential and EMT of PTC cells, both in vitro and in vivo. Mechanistically, CLIP170KD triggered the activation of the TGF-β pathway, subsequently promoting tumor cell migration, invasion, and EMT. Remarkably, the TGF-β inhibitor LY2157299 effectively countered TGF-β activity and significantly reversed tumor metastasis and EMT induced by CLIP170 knockdown. In summary, these findings collectively propose CLIP170 as a promising therapeutic target to mitigate metastatic tendencies in PTC.

SpCTP3 from the hyperaccumulator Sedum plumbizincicola positively regulates cadmium tolerance by interacting with SpMDH1

Cadmium (Cd) is a heavy metal pollutant mainly originating from the discharge of industrial sewage, irrigation with contaminated water, and the use of fertilizers. The phytoremediation of Cd polluted soil depends on the identification of the associated genes in hyperaccumulators. Here, a novel Cd tolerance gene (SpCTP3) was identified in hyperaccumulator Sedum plumbizincicola. The results of Cd2+ binding and thermodynamic analyses, revealed the CXXC motif in SpCTP3 functions is a Cd2+ binding site. A mutated CXXC motif decreased binding to Cd by 59.93%. The subcellular localization analysis suggested that SpCTP3 is primarily a cytoplasmic protein. Additionally, the SpCTP3-overexpressing (OE) plants were more tolerant to Cd and accumulated more Cd than wild-type Sedum alfredii (NHE-WT). The Cd concentrations in the cytoplasm of root and leaf cells were significantly higher (53.75% and 71.87%, respectively) in SpCTP3-OE plants than in NHE-WT. Furthermore, malic acid levels increased and decreased in SpCTP3-OE and SpCTP3-RNAi plants, respectively. Moreover, SpCTP3 interacted with malate dehydrogenase 1 (MDH1). Thus, SpCTP3 helps regulate the subcellular distribution of Cd and increases Cd accumulation when it is overexpressed in plants, ultimately Cd tolerance through its interaction with SpMDH1. This study provides new insights relevant to improving the Cd uptake by Sedum plumbizincicola.

NIR-responsive carbon dots as an oxidative-stress amplifier and hyperthermia-induced superior photothermal in-vitro anticancer activity

A facile wet chemistry-based strategy was developed to synthesize orange emissive Carbon dots (O-CDs) based on dehydration-induced ring-fusion of the precursor (1,3-dihydroxynaphthalene) in a dehydrating sulfuric acid medium. The O-CDs revealed significant Near-infrared (808nm) light harvesting potential with outstanding photothermal conversion efficiency (~58%) and hence exhibited excellent NIR-light stimulated photothermal anticancer performance. Furthermore, these fluorescent O-CD nanoprobes displayed excitation-dependent polychromatic emissions in the range of 540–640nm, with the dual emission maxima at 540 and 580nm, corresponding to yellow and orange emission at the excitation of 490nm. Various mitochondrial, as well as cytoplasmic apoptosis-related proteins and genes were upregulated during photothermal treatment, which suggests that hyperthermia and oxidative stress have a major impact on cell death. The anticancer activity of O-CDs is likely due to the elevated reactive oxygen species (ROS) amplification in cooperation with the hyperthermia effect. This study offers a potential alternative to bio-nanomedicine for cancer treatment by carbon-based photothermal therapy.

Phylogenetic Analysis of Wall-Associated Kinase Genes in Triticum Species and Characterization of TaWAK7 Involved in Wheat Powdery Mildew Resistance.

Wall-associated kinases (WAKs), a group of receptor-like kinases, have been found to play important roles in defending against pathogens and in various developmental processes. However, the importance of this family in wheat remains largely unknown. Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), which initiates infection on the cell surface and forms haustoria inside the cell; therefore, the defense to Bgt involves extracellular and subsequently intracellular signals. In this study, WAKs were identified genome-wide and analyzed phylogenetically, and then a transmembrane WAK gene that putatively participated in pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity to Bgt was functionally and evolutionarily investigated. In total, 1,193 WAKs were identified from wheat and its Gramineae relatives. Phylogenetic analysis indicated that WAKs expanded through tandem duplication or segment duplication. TaWAK7, from chromosome 2A, was identified as a Bgt-inducible gene both in susceptible and resistant materials, but it showed distinct responsive patterns. Functional analysis showed that TaWAK7 was involved in both the basal and resistance gene-mediated resistances. The specific gene structures and protein characteristics of TaWAK7, along with its orthologs, were characterized both in subgenomes of Triticum spp. and in the A genome of multiple wheat accessions, which revealed that TaWAK7 orthologs underwent complex evolution with frequent gene fusion and domain deletion. In addition, three cytoplasmic proteins interacting with TaWAK7 were indicated by yeast two-hybrid and bimolecular fluorescence complementation assays. Binding of TaWAK7 with these proteins could change its subcellular localization from the plasma membrane to the cytoplasm. This study provides a better understanding of the evolution of WAKs at the genomic level and TaWAK7 at the gene level and provides useful clues for further investigation of how WAKs transmit the extracellular signals to the cytoplasm to activate defense responses.

The heme binding protein ChuX is a regulator of heme degradation by the ChuS protein in Escherichia coli O157:H7

Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes.

UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope.

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE.

Development of FRET Biosensor to Characterize CSK Subcellular Regulation.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

Silymarin and MSC-exosomes ameliorate thioacetamide-evoked renal fibrosis by inhibiting TGF-β/SMAD pathway in rats

BackgroundTGF-β1 and SMAD3 are particularly pathogenic in the progression of renal fibrosis.AimThis study aimed to evaluate the kidney protective potentials of silymarin (SM) and exosomes of mesenchymal stem cells against the nephrotoxin thioacetamide (TAA) in rats.Methods32 female rats were randomly assigned into four groups: the control group, the TAA group, the TAA + SM group, and the TAA + Exosomes group. The kidney homogenates from all groups were examined for expression levels of TGF-β receptors I and II using real-time PCR, expression levels of collagen type I and CTGF proteins using ELISA, and the expression levels of nuclear SMAD2/3/4, cytoplasmic SMAD2/3, and cytoplasmic SMAD4 proteins using the western blot technique.ResultsCompared to the control group, the injection of TAA resulted in a significant increase in serum levels of urea and creatinine, gene expression levels of TβRI and TβRII, protein expression levels of both collagen I and CTGF proteins, cytoplasmic SMAD2/3 complex, and nuclear SMAD2/3/4 (p-value < 0.0001), with significantly decreased levels of the co-SMAD partner, SMAD4 (p-value < 0.0001). Those effects were reversed considerably in both treatment groups, with the superiority of the exosomal treatment regarding the SMAD proteins and the expression levels of the TβRI gene, collagen I, and CTGF proteins returning to near-control values (p-value > 0.05).ConclusionUsing in vitro and in vivo experimental approaches, the research discovered a reno-protective role of silymarin and exosomes of BM-MSCs after thioacetamide-induced renal fibrosis in rats, with the advantage of exosomes.

Post-Translational Regulation of a Bidomain Glycerol-3-Phosphate Dehydrogenase Catalyzing Glycerol Synthesis under Salinity Stress in Chlamydomonas reinhardtii

Core chlorophytes possess glycerol-3-phosphate dehydrogenases (GPDs) with an unusual bidomain structure, consisting of a glycerol-3-phosphate phosphatase (GPP) domain fused to canonical GPD domains. These plastid-localized enzymes have been implicated in stress responses, being required for the synthesis of glycerol under high salinity and triacylglycerols under nutrient deprivation. However, their regulation under varying environmental conditions is poorly understood. C. reinhardtii transgenic strains expressing constitutively bidomain GPD2 did not accumulate glycerol or triacylglycerols in the absence of any environmental stress. Although the glycerol contents of both wild type and transgenic strains increased significantly upon exposure to high salinity, cycloheximide, an inhibitor of cytoplasmic protein synthesis, abolished this response in the wild type. In contrast, GPD2 transgenic strains were still capable of glycerol accumulation when cultured in medium containing cycloheximide and NaCl. Thus, the pre-existing GPD2 protein appears to become activated for glycerol synthesis upon salt stress. Interestingly, staurosporine, a non-specific inhibitor of protein kinases, prevented this post-translational GPD2 protein activation. Structural modeling analyses suggested that substantial conformational rearrangements, possibly triggered by high salinity, may characterize an active GPD2 GPP domain. Understanding this mechanism(s) may provide insights into the rapid acclimation responses of microalgae to osmotic/salinity stress.

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM)

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins.In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart.To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

Signaling of Plant Defense Mediated by Receptor-like Kinases, Receptor-like Cytoplasmic Protein Kinases and MAPKs Triggered by Fungal Chitin in Horticultural Crops

Fresh horticultural products are economically significant foods that are highly demanded by consumers worldwide; however, they are highly perishable and susceptible to deterioration by fungi, which contribute to their short shelf-life and cause significant post-harvest losses. Among the alternatives suggested for fungal control in plants is the elicitation of the innate plant defense mechanism, which may be activated when specific molecules of the phytopathogen, such as chitin, are recognized. Chitin is a long-chain polymer of N-acetyl-α-D-glucosamine of the fungal cell wall; it possesses biological activity by eliciting the plant immune response. This molecule and its oligosaccharides are recognized through transmembrane receptors known as receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Mediated by receptor-like cytoplasmic kinases (RLCKs), which bind to the intracellular domain of these receptors, they initiate intracellular signal transduction via MAP kinases, triggering the plant defense response. In model plants, such as Oryza sativa (rice) and Arabidopsis thaliana, the set of RLK/RLP-RLCK-MAP kinases is involved in plant immunity triggered by chitin. Furthermore, in horticultural products, research into the molecular events between these three elements has suggested that similar processes occur. However, little is known about these molecular events in fruits. Against this background, the present review provides the most recent and relevant findings on the molecular associations of these three elements in the response to fungal chitin in plants and outlines which elements could participate in this signaling process in horticultural crops.

Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways.

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

Cytoplasmic Leakage and Protein Leakage Analysis of Ocimum Gratissimum Stem Extract-Mediated Silver Nanoparticles Against Wound Pathogens

ABSTRACT Aim: The current work intends to examine the antibacterial activity of silver nanoparticles (AgNPs) mediated by Ocimum gratissimum stem extract against wound infections. Materials and Methods: To evaluate the membrane damage brought on by AgNPs, analyses of cytoplasmic leakage and protein leakage assays were performed. Results: The outcomes demonstrated that all of the tested bacterial strains were significantly resistant to the AgNPs’ antibacterial activity. AgNPs damaged membranes and caused cellular contents to leak in the target pathogens, according to an examination of protein and cytoplasmic leakage. Conclusion: According to the current investigation, AgNPs mediated by Ocimum gratissimum stem extract may be effective antibacterial agents against microorganisms that cause wounds.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

Scaffolds and the scaffolding domain: an alternative paradigm for caveolin-1 signaling.

Caveolin-1 (Cav1) is a 22 kDa intracellular protein that is the main protein constituent of bulb-shaped membrane invaginations known as caveolae. Cav1 can be also found in functional non-caveolar structures at the plasma membrane called scaffolds. Scaffolds were originally described as SDS-resistant oligomers composed of 10-15 Cav1 monomers observable as 8S complexes by sucrose velocity gradient centrifugation. Recently, cryoelectron microscopy (cryoEM) and super-resolution microscopy have shown that 8S complexes are interlocking structures composed of 11 Cav1 monomers each, which further assemble modularly to form higher-order scaffolds and caveolae. In addition, Cav1 can act as a critical signaling regulator capable of direct interactions with multiple client proteins, in particular, the endothelial nitric oxide (NO) synthase (eNOS), a role believed by many to be attributable to the highly conserved and versatile scaffolding domain (CSD). However, as the CSD is a hydrophobic domain located by cryoEM to the periphery of the 8S complex, it is predicted to be enmeshed in membrane lipids. This has led some to challenge its ability to interact directly with client proteins and argue that it impacts signaling only indirectly via local alteration of membrane lipids. Here, based on recent advances in our understanding of higher-order Cav1 structure formation, we discuss how the Cav1 CSD may function through both lipid and protein interaction and propose an alternate view in which structural modifications to Cav1 oligomers may impact exposure of the CSD to cytoplasmic client proteins, such as eNOS.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Alterations in HCN1 expression and distribution during epileptogenesis in rats

BackgroundThe hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear. ObjectiveThe aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis. MethodsWe utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques. ResultsIn the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression. ConclusionsAlterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.

Quantitative analysis of the spatial distance between autophagy-related membrane structures and the endoplasmic reticulum in Saccharomyces cerevisiae

ABSTRACT Macroautophagy/autophagy is the process by which cells degrade their cytoplasmic proteins or organelles in vacuoles to maintain cellular homeostasis under severe environmental conditions. In the yeast Saccharomyces cerevisiae, autophagy-related (Atg) proteins essential for autophagosome formation accumulate near the vacuole to form the dot-shaped phagophore assembly site/pre-autophagosomal structure (PAS). The PAS then generates the phagophore/isolation membrane (PG), which expands to become a closed double-membrane autophagosome. Hereinafter, we refer to the PAS, PG, and autophagosome as autophagy-related structures (ARSs). During autophagosome formation, Atg2 is responsible for tethering the ARS to the endoplasmic reticulum (ER) via ER exit sites (ERESs), and for transferring phospholipids from the ER to ARSs. Therefore, ARS and the ER are spatially close in the presence of Atg2 but are separated in its absence. Because the contact of an ARS with the ER must be established at the earliest stage of autophagosome formation, it is important to know whether the ARS is tethered to the ER. In this study, we developed a rapid and objective method to estimate tethering of the ARS to the ER by measuring the distance between the ARS and ERES under fluorescence microscopy, and found that tethering of the ARS to the ER was lost without Atg1. This method might be useful to predict the tethering activity of Atg2. Abbreviation: ARS, autophagy-related structure; Dautas, automated measurement of the distance between autophagy-related structures and ER exit sites analysis system; ERES, endoplasmic reticulum exit site; PAS, phagophore assembly site/pre-autophagosomal structure; PCR, polymerase chain reaction; PG, phagophore/isolation membrane; prApe1, precursor of vacuolar aminopeptidase I; Qautas, quantitative autophagy-related structure analysis system; SD/CA; synthetic dextrose plus casamino acid medium; WT, wild-type