Golgi protein ACBD3 downregulation sensitizes cells to ferroptosis.

Ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, is emerging as a promising target in cancer therapy. It is regulated by a network of molecules and pathways that modulate lipid metabolism, iron homeostasis and redox balance, and related processes. However, there are still numerous regulatory molecules intricately involved in ferroptosis that remain to be identified. Here, we indicated that suppression of Golgi protein acyl-coenzyme A binding domain A containing 3 (ACBD3) increased the sensitivity of Henrieta Lacks and PANC1 cells to ferroptosis. ACBD3 knockdown increases labile iron levels by promoting ferritinophagy. This increase in free iron, coupled with reduced levels of glutathione peroxidase 4 due to ACBD3 knockdown, leads to the accumulation of reactive oxygen species and lipid peroxides. Moreover, ACBD3 knockdown also results in elevated levels of polyunsaturated fatty acid-containing glycerophospholipids through mechanisms that remain to be elucidated. Furthermore, inhibition of ferrtinophagy in ACBD3 downregulated cells by knocking down the nuclear receptor co-activator 4 or Bafilomycin A1 treatment impeded ferroptosis. Collectively, our findings highlight the pivotal role of ACBD3 in governing cellular resistance to ferroptosis and suggest that pharmacological manipulation of ACBD3 levels is a promising strategy for cancer therapy.

ACAP4 interacts with CrkII to promote the recycling of integrin ß1.

ACAP4, a GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (ARF6), plays import roles in cell migration, cell polarity, vesicle trafficking and tumorigenesis. Similarly, the ubiquitously expressed adaptor protein CrkII functions in a wide range of cellular activities, including cell proliferation, T cell adhesion and activation, tumorigenesis, and bacterial pathogenesis. Here, we demonstrate that ACAP4 physically interacts with CrkII. Biochemical experiments revealed that ACAP4550-660 and the SH3N domain of CrkII are responsible for the interaction. Functional characterization showed that the interaction is required for the recruitment of ACAP4 to the plasma membrane where ACAP4 functions to regulate the recycling of the signal transducer integrin ß1. Thus, we suggest that the CrkII-ACAP4 complex may be involved in regulation of cell adhesion.

MST4 kinase phosphorylates ACAP4 protein to orchestrate apical membrane remodeling during gastric acid secretion.

Digestion in the stomach depends on acidification of the lumen. Histamine-elicited acid secretion is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. Our recent study revealed the functional role of PKA-MST4-ezrin signaling axis in histamine-elicited acid secretion. However, it remains uncharacterized how the PKA-MST4-ezrin signaling axis operates the insertion of H,K-ATPases into the apical plasma membranes of gastric parietal cells. Here we show that MST4 phosphorylates ACAP4, an ARF6 GTPase-activating protein, at Thr545 Histamine stimulation activates MST4 and promotes MST4 interaction with ACAP4. ACAP4 physically interacts with MST4 and is a cognate substrate of MST4 during parietal cell activation. The phosphorylation site of ACAP4 by MST4 was mapped to Thr545 by mass spectrometric analyses. Importantly, phosphorylation of Thr545 is essential for acid secretion in parietal cells because either suppression of ACAP4 or overexpression of non-phosphorylatable ACAP4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, persistent overexpression of MST4 phosphorylation-deficient ACAP4 results in inhibition of gastric acid secretion and blockage of tubulovesicle fusion to the apical membranes. Significantly, phosphorylation of Thr545 enables ACAP4 to interact with ezrin. Given the location of Thr545 between the GTPase-activating protein domain and the first ankyrin repeat, we reason that MST4 phosphorylation elicits a conformational change that enables ezrin-ACAP4 interaction. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ACAP4 signaling cascade to polarized acid secretion in gastric parietal cells.

Extraction and characterization of nanocellulose from cattail leaves: Morphological, microstructural and thermal properties.

Hydrogen peroxide combined with acid treatment demonstrates its respective characteristics for the separation of lignocellulosic biomass. Herein, holocellulose was extracted from Cattail leaves (CL) by a two-step treatment with alkali and hydrogen peroxide-acetic acid (HPAA). Then carboxylated nanocellulose was hydrolyzed with a mixed organic/inorganic acid. The chemical composition of the holocellulose and the physicochemical properties of the separated carboxylated nanocellulose were comparable. Carboxyl groups were introduced on the nanocellulose as a result of the esterification process with citric acid (CA), which endows the nanocellulose with high thermal stability (315-318 °C) and good light transmission (>80 %). Furthermore, morphological analyses revealed that nanocellulose had a spider-web-like structure with diameter between 5 and 20 nm.

Cellulose separation from ramie bone by one step process with green hydrogen peroxide-citric acid.

Ramie bone (RB), an agricultural waste generated in the textile industry, is a vastly productive renewable natural resource with the potential to be used as a source of cellulose. In this study, ramie bone cellulose (RB-CE) was obtained in one step using a simple and ecologically friendly hydrogen peroxide-citric acid (HPCA) treatment procedure that avoided the use of halogenated reagents and strong acids while also streamlining the treatment processes. Various analytical methods were used to investigate the chemical composition and structure, crystallinity, morphology, thermal properties, surface area and hydration properties of cellulose separated at different treatment temperatures. HPCA successfully removed lignin and hemicellulose from RB, according to chemical composition analysis and FTIR. RB-CE had a type I cellulose crystal structure, and the crystallinity improved with increasing treatment temperature, reaching 72.51 % for RB-CE90. The RB-CE showed good thermal stability with degradation temperatures ranging from 294.2 °C to 319.1 °C. Furthermore, RB-CE had a high water/oil binding capacity, with RB-CE90 having WHC and OBC of 9.68 g/g and 7.24 g/g, respectively. The current work serves as a model for the environmentally friendly and convenient extraction of cellulose from biomass, and the cellulose obtained can be employed in the field of food and composite materials.

Application of 3 different types of pedicled adipofascial flaps in the repair of deep dead space wounds.

INTRODUCTION: Dead space is an important risk factor for poor wound healing; therefore, it is important to effectively fill deep dead space through individualized tissue flap design during the repair of complex wounds. Adipofascial flaps have yielded good results in the repair of deep dead space wounds. OBJECTIVE: The authors evaluated the efficacy of 3 kinds of adipofascial flaps to repair deep dead space wounds. METHODS: From January 2019 to January 2022, 15 patients with complicated wounds accompanied by deep dead space underwent repair via 1 of 3 kinds of adipofascial flaps, and the clinical efficacy was observed. RESULTS: All 15 transplanted adipofascial flaps exhibited complete survival, and within a mean follow-up of 14.7 months, both the donor and recipient sites had successfully healed. CONCLUSION: The traditional pedicled adipofascial flap was used to repair single deep dead space wounds, and pedicled perforator adipofascial extension flaps or layered fasciocutaneous flaps were used on compound tissue defect wounds, thus providing a relatively simple, safe, and effective method to repair a small area of tissue defect with deep dead space wounds.

ACAP4 protein cooperates with Grb2 protein to orchestrate epidermal growth factor-stimulated integrin ß1 recycling in cell migration.

ARF6 GTPase is an important regulator of membrane trafficking and actin-based cytoskeleton dynamics active at the leading edge of migrating cells. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Our recent proteomic analyses of ARF6 effectors have identified a novel ARF6 GTPase-activating protein, ACAP4, essential for EGF-induced cell migration. However, molecular mechanisms underlying ACAP4-mediated cell migration have remained elusive. Here, we show that ACAP4 regulates integrin ß1 dynamics during EGF-stimulated cell migration by interaction with Grb2. Our biochemical study shows that EGF stimulation induces phosphorylation of tyrosine 733, which enables ACAP4 to bind Grb2. This interaction of ACAP4 with Grb2 regulates integrin ß1 recycling to the plasma membrane. Importantly, knockdown of ACAP4 by siRNA or overexpression of ACAP4 decreased recycling of integrin ß1 to the plasma membrane and reduced integrin-mediated cell migration. Taken together, these results suggest a novel function for ACAP4 in the regulation of cell migration through controlling integrin ß1 dynamics.

Structural basis of wedging the Golgi membrane by FAPP pleckstrin homology domains.

The mechanisms underlying Golgi targeting and vesiculation are unknown, although the responsible phosphatidylinositol 4-phosphate (PtdIns(4)P) ligand and four-phosphate-adaptor protein (FAPP) modules have been defined. The micelle-bound structure of the FAPP1 pleckstrin homology domain reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. Mutations compromising the exposed hydrophobicity of full-length FAPP2 abolish lipid monolayer binding and compression. The trafficking process begins with an electrostatic approach, phosphoinositide sampling and perpendicular penetration. Extensive protein contacts with PtdIns(4)P and neighbouring phospholipids reshape the bilayer and initiate tubulation through a conserved wedge with features shared by diverse protein modules.

Golgi protein FAPP2 tubulates membranes.

The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network.

ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity.

Glycosphingolipid (GSL) metabolism is involved in various physiological processes, including all major cell signaling pathways, and its dysregulation is linked to some diseases. The four-phosphate adaptor protein FAPP2-mediated glucosylceramide (GlcCer) transport for complex GSL synthesis has been studied extensively. However, the molecular machinery of FAPP2 as a GlcCer-transferring protein remains poorly defined. Here, we identify a Golgi-resident protein, acyl-coenzyme A binding domain containing 3 (ACBD3), as an interacting partner of FAPP2. We find that ACBD3 knockdown leads to dramatic Golgi fragmentation, which subsequently causes FAPP2 dispersal throughout the cytoplasm and a decreased localization at trans-Golgi network. The further quantitative lipidomic analysis indicates that ACBD3 knockdown triggers abnormal sphingolipid metabolism. Interestingly, the expression of siRNA-resistant full-length ACBD3 can rescue these defects caused by ACBD3 knockdown. These data reveal critical roles for ACBD3 in maintaining the integrity of Golgi morphology and cellular sphingolipid homeostasis and establish the importance of the integrated Golgi complex for the transfer of GlcCer and complex GSL synthesis.

The J-domain cochaperone Rsp1 interacts with Mto1 to organize noncentrosomal microtubule assembly.

Microtubule biogenesis initiates at various intracellular sites, including the centrosome, the Golgi apparatus, the nuclear envelope, and preexisting microtubules. Similarly, in the fission yeast Schizosaccharomyces pombe, interphase microtubules are nucleated at the spindle pole body (SPB), the nuclear envelope, and preexisting microtubules, depending on Mto1 activity. Despite the essential role of Mto1 in promoting microtubule nucleation, how distribution of Mto1 in different sites is regulated has remained elusive. Here, we show that the J-domain cochaperone Rsp1 interacts with Mto1 and specifies the localization of Mto1 to non-SPB nucleation sites. The absence of Rsp1 abolishes the localization of Mto1 to non-SPB nucleation sites, with concomitant enrichment of Mto1 to the SPB and the nuclear envelope. In contrast, Rsp1 overexpression impairs the localization of Mto1 to all microtubule organization sites. These findings delineate a previously uncharacterized mechanism in which Rsp1-Mto1 interaction orchestrates non-SPB microtubule formation.

Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world and metastasis is the leading cause of death associated with HCC. Hypoxia triggers the epithelial-mesenchymal transition (EMT) of cancer cells, which enhances their malignant character and elevates metastatic risk. Supervillin associates tightly with the membrane and cytoskeleton, promoting cell motility, invasiveness, and cell survival. However, the roles of supervillin in HCC metastasis remain unclear. METHODS: Tissue microarray technology was used to immunohistochemically stain for supervillin antibody in 173 HCC tissue specimens and expression levels correlated with the clinicopathological variables. Tumor cell motility and invasiveness, as well as changes in the mRNA expression levels of genes associated with cancer cell EMT, were investigated. The relationship between supervillin and Rho GTPases was examined using Co-IP and GST pull-down. RESULTS: Hypoxia-induced upregulation of supervillin promoted cancer cell migration and invasion via the activation of the ERK/p38 pathway downstream of RhoA/ROCK signaling. Furthermore, supervillin regulated the expression of EMT genes during hypoxia and accelerated the metastasis of HCC in vivo. CONCLUSIONS: Hypoxia-induced increase in supervillin expression is a significant and independent predictor of cancer metastasis, which leads to poor survival in HCC patients. Our results suggest that supervillin may be a candidate prognostic factor for HCC and a valuable target for therapy.

Novel spider-web-like nanoporous networks based on jute cellulose nanowhiskers.

Cellulose nanowhiskers as a kind of renewable and biocompatible nanomaterials evoke much interest because of its versatility in various applications. Herein, for the first time, a novel controllable fabrication of spider-web-like nanoporous networks based on jute cellulose nanowhiskers (JCNs) deposited on the electrospun (ES) nanofibrous membrane by simple directly immersion-drying method is reported. Jute cellulose nanowhiskers were extracted from jute fibers with a high yield (over 80%) via a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)/NaBr/NaClO system selective oxidization combined with mechanical homogenization. The morphology of JCNs nanoporous networks/ES nanofibrous membrane architecture, including coverage rate, pore-width and layer-by-layer packing structure of the nanoporous networks, can be finely controlled by regulating the JCNs dispersions properties and drying conditions. The versatile nanoporous network composites based on jute cellulose nanowhiskers with ultrathin diameters (3-10 nm) and nanofibrous membrane supports with diameters of 100-300 nm, would be particularly useful for filter applications.

In situ growth of silver nanoparticles on TEMPO-oxidized jute fibers by microwave heating.

Cellulose fibers deposited with metallic nanoparticles as one kind of renewable, biocompatible and antimicrobial nanomaterials evoke much interest because of their versatility in various applications. Herein, for the first time, a facile, simple and rapid method was developed to fabricate TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) selectively oxidized jute fibers in situ deposited with silver nanoparticles in the absence of reducing reagents. The average size of silver nanoparticles deposited on the fibers is 50.0 ± 2.0 nm by microwave heating for 5 min and 90.0 ± 4.7 nm for 10 min heating sample, respectively. The versatile jute-silver nanoparticles nanocomposites with superior thermal stability and high crystallinity would be particularly useful for applications in the public health care and biomedical fields.

Polarized sorting and trafficking in epithelial cells.

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.

Cellulose nanowhiskers extracted from TEMPO-oxidized jute fibers.

Cellulose nanowhiskers is a kind of renewable and biocompatible nanomaterials evoke much interest because of its versatility in various applications. Here, for the first time, a novel controllable fabrication of cellulose nanowhiskers from jute fibers with a high yield (over 80%) via a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)/NaBr/NaClO system selective oxidization combined with mechanical homogenization is reported. The versatile jute cellulose nanowhiskers with ultrathin diameters (3-10 nm) and high crystallinity (69.72%), contains C6 carboxylate groups converted from C6 primary hydroxyls, which would be particularly useful for applications in the nanocomposites as reinforcing phase, as well as in tissue engineering, pharmaceutical and optical industries as additives.

PKA-mediated protein phosphorylation regulates ezrin-WWOX interaction.

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton, and also participate in signal-transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies have mapped the PKA-mediated phosphorylation site to Ser(66) and established its functional role in parietal cell activation [R. Zhou et al., Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651-35659], but the underlying basis for this regulation is not known. Here, we provide the first evidence that PKA-mediated phosphorylation of Ser(66)regulates the interaction of ezrin with WWOX, a WW domain-containing protein. Our biochemical study reveals that ezrin directly binds to the first WW domain of WWOX via its C-terminal tyrosine-containing polyproline sequence (470)PPPPPPVY(477). Mutational analyses further demonstrate that tyrosine(477) is essential for the ezrin-WWOX interaction. In addition, our study shows that PKA-mediated phosphorylation of ezrin is essential and sufficient for the apical localization of WWOX protein as disruption of ezrin-WWOX interaction eliminated the apical localization of WWOX. Finally, our study demonstrates the essential role of ezrin-WWOX interaction in the apical membrane remodeling associated with H,K-ATPase recruitment. Taken together, these results define a novel molecular mechanism underlying phospho-regulation of ezrin function by PKA in parietal cell activation.

PKA-mediated protein phosphorylation protects ezrin from calpain I cleavage.

Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase (PKA) activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies demonstrate the functional relevance of PKA-mediated phosphorylation of ezrin in parietal cell secretion [R. Zhou, X. Cao, C. Watson, Y. Miao, Z. Guo, J.G. Forte, X. Yao, Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651]. Here we show that activation of PKA protects ezrin from calpain I-mediated proteolysis without alteration of calpain I activation and fodrin breakdown. To determine whether phosphorylation of Ser66 by PKA affects the insensitivity to the calpain I-mediated cleavage, recombinant proteins of ezrin, both wild type and S66A/D mutants, were incubated with the purified calpain I. Indeed, phosphorylation-like S66D mutant ezrin is resistant to calpain I-mediated proteolysis while wild type and S66A mutant were sensitive. In fact, expression of phosphorylation-like S66D, but not S66A, mutant in parietal cells confers its resistance to calpain I-mediated proteolysis. Taken together, these results indicate that phosphorylation of ezrin by PKA modulates its sensitivity to calpain I cleavage.

PALS1 specifies the localization of ezrin to the apical membrane of gastric parietal cells.

The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.

Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation.

Gastric ezrin was initially identified as a phosphoprotein associated with parietal cell activation. To explore the nature of ezrin phosphorylation, proteins from resting and secreting gastric glands were subjected to two-dimensional SDS-PAGE. Histamine triggers acid secretion and a series of acidic isoforms of ezrin on two-dimensional SDS-PAGE. Mass spectrometric analysis of these acidic ezrin spots induced by stimulation suggests that Ser66 is phosphorylated. To determine whether Ser66 is a substrate of protein kinase A (PKA), recombinant proteins of ezrin, both wild type and S66A mutant, were incubated with the catalytic subunit of PKA and [32P]ATP. Incorporation of 32P into wild type but not the mutant ezrin verified that Ser66 is a substrate of PKA. In addition, expression of S66A mutant ezrin in cultured parietal cells attenuates the dilation of apical vacuolar membrane associated with stimulation by histamine, indicating that PKA-mediated phosphorylation of ezrin is necessary for acid secretion. In fact, expression of phosphorylation-like S66D mutant in parietal cells mimics histamine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution revealed a blockade of stimulation-induced proton pump mobilization in S66A but not S66D ezrin-expressing parietal cells. These data suggest that PKA-mediated phosphorylation of ezrin plays an important role in mediating the remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells.