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Acta Biomaterialia May 2024The Golgi apparatus (GA) is a vital target for anticancer therapy due to its sensitivity against reactive oxygen species (ROS)-induced oxidative stress that could lead...
The Golgi apparatus (GA) is a vital target for anticancer therapy due to its sensitivity against reactive oxygen species (ROS)-induced oxidative stress that could lead to cell death. In this study, we designed a series of aggregation-induced emission (AIE)-based photosensitizers (TPAPyTZ, TPAPyTC, TPAPyTM, and TPAPyTI) carrying different ROS with selective GA-targeted ability. The in vitro study showed that TPAPyTZ and TPAPyTC displayed strong AIE characteristics, robust type-I/II ROS production capabilities, specific GA-targeted, high photostability, and high imaging quality. The cell-uptake of TPAPyTZ was found primarily through an energy-dependent caveolae/raft-mediated endocytosis pathway. Remarkably, TPAPyTZ induced GA-oxidative stress, leading to GA fragmentation, downregulation of GM130 expression, and activation of mitochondria caspase-related apoptosis during photodynamic therapy (PDT). In vivo experiments revealed that TPAPyTZ significantly inhibited tumor proliferation under lower-intensity white light irradiation with minimal side effects. Overall, our work presents a promising strategy for designing AIEgens for fluorescence imaging-guided PDT. Additionally, it enriched the collection of GA-targeted leads for the development of cancer theranostics capable of visualizing dynamic changes in the GA during cancer cell apoptosis, which could potentially enable early diagnosis applications in the future. STATEMENT OF SIGNIFICANCE: AIE luminogens (AIEgens) are potent phototheranostic agents that can exhibit strong fluorescence emission and enhance ROS production in the aggregate states. In this study, through the precise design of photosensitizers with four different electron-acceptors, we constructed a series of potent AIEgens (TPAPyTZ, TPAPyTC, TPAPyTM, and TPAPyTI) with strong fluorescence intensity and ROS generation capacity. Among them, TPAPyTZ with an extended π-conjugation displayed the strongest ROS generation ability and anti-tumor activity, resulting in an 88 % reduction in tumor weight. Our studies revealed that the enhanced activity of TPAPyTZ may be due to its unique Golgi apparatus (GA)-targeted ability, which causes GA oxidative stress followed by effective cancer cell apoptosis. This unique GA-targeted feature of TPAPyTZ remains rare in the reported AIEgens, which mainly target organelles such as lysosome, mitochondria, and cell membrane. The successful design of a GA-targeted and potent AIEgen could enrich the collection of GA-targeted luminogens, providing a lead theranostic for the further development of fluorescence imaging-guided PDT, and serving as a tool to explore the potential mechanism and discover new GA-specific drug targets.
PubMed: 38801870
DOI: 10.1016/j.actbio.2024.05.034 -
Biotechnology Journal May 2024Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include...
High-activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane.
Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (k), a 137% decrease in the Michaelis constant (K), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6 ± 20 mg L with a specific activity of 96 ± 0.32 U mg, resulting in a total enzyme activity of 19137 ± 1131 U L, demonstrating significant potential for industrial applications.
Topics: Humans; Recombinant Proteins; Protein Engineering; Carboxypeptidase B; Cell Membrane; Golgi Apparatus; Saccharomycetales; Mutation; Pichia; Protein Sorting Signals; Protein Transport
PubMed: 38797728
DOI: 10.1002/biot.202400098 -
Nature Communications May 2024To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient...
To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2 mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.
Topics: Animals; Male; Mice; Carnitine O-Palmitoyltransferase; Cholesterol; Fasting; Golgi Apparatus; Hepatocytes; Homeostasis; Lipid Metabolism; Liver; Mice, Inbred C57BL; Mice, Knockout; rab GTP-Binding Proteins; Triglycerides
PubMed: 38796472
DOI: 10.1038/s41467-024-48959-x -
Journal of Controlled Release :... Jul 2024Nutrient or energy deprivation, especially glucose restriction, is a promising anticancer therapeutic approach. However, establishing a precise and potent deprivation...
Nutrient or energy deprivation, especially glucose restriction, is a promising anticancer therapeutic approach. However, establishing a precise and potent deprivation strategy remains a formidable task. The Golgi morphology is crucial in maintaining the function of transport proteins (such as GLUT1) driving glycolysis. Thus, in this study, we present a "Golgi-customized Trojan horse" based on tellurium loaded with apigenin (4',5,7-trihydroxyflavone) and human serum albumin, which was able to induce GLUT1 plasma membrane localization disturbance via Golgi dispersal leading to the inhibition of tumor glycolysis. Diamond-shaped delivery system can efficiently penetrate into cells as a gift like Trojan horse, which decomposes into tellurite induced by intrinsically high HO and GSH levels. Consequently, tellurite acts as released warriors causing up to 3.8-fold increase in Golgi apparatus area due to the down-regulation of GOLPH3. Further, this affects GLUT1 membrane localization and glucose transport disturbance. Simultaneously, apigenin hinders ongoing glycolysis and causes significant decrease in ATP level. Collectively, our "Golgi-customized Trojan horse" demonstrates a potent antitumor activity because of its capability to deprive energy resources of cancer cells. This study not only expands the applications of tellurium-based nanomaterials in the biomedicine but also provides insights into glycolysis restriction for anticancer therapy.
Topics: Humans; Glycolysis; Golgi Apparatus; Cell Membrane; Glucose Transporter Type 1; Apigenin; Tellurium; Cell Line, Tumor; Neoplasms; Animals; Antineoplastic Agents; Glucose
PubMed: 38789089
DOI: 10.1016/j.jconrel.2024.05.025 -
Bioorganic Chemistry Jul 2024Depression is a debilitating mental illness that poses a serious threat to human health. Nitric Oxide (NO), as an important gasotransmitter, is closely associated with...
Depression is a debilitating mental illness that poses a serious threat to human health. Nitric Oxide (NO), as an important gasotransmitter, is closely associated with the pathogenesis of depressive disorders. Effective monitoring of NO fluctuation is beneficial for the diagnosis of depression and therapy assessment of antidepressants. Currently, there is a lack of effective methods for rapidly and sensitively identifying NO and elucidating its relationship with depression diseases. Herein, we developed a NIR dye TJ730-based fluorescent probe TJ730-Golgi-NO incorporating benzenesulfonamide as a Golgi-targeted moiety and the thiosemicarbazide group for NO detection. The probe exhibited turn-on fluorescence ability and a large Stokes shift of 158 nm, which shows high sensitivity, selectivity, and rapid response (<1 min) for NO detection. TJ730-Golgi-NO could detect exogenous and endogenous NO in cells stimulated by Glu and LPS, and target Golgi apparatus. Moreover, we disclose a significant increase of NO in the depression model and a weak fluorescence evidenced in the fluoxetine-treated depression mice. This study provides a competent tool for studying the function of NO and helping improve the effective treatment of depression diseases.
Topics: Fluorescent Dyes; Animals; Nitric Oxide; Mice; Golgi Apparatus; Depression; Molecular Structure; Humans; Disease Models, Animal; Male; Structure-Activity Relationship; Infrared Rays; Dose-Response Relationship, Drug; Optical Imaging; RAW 264.7 Cells
PubMed: 38788368
DOI: 10.1016/j.bioorg.2024.107476 -
Life Science Alliance Aug 2024Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as...
Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.
Topics: Humans; Membrane Proteins; Cholesterol; Ubiquitin-Protein Ligases; Endoplasmic Reticulum; HeLa Cells; Golgi Apparatus; Secretory Pathway; Protein Binding; Nerve Tissue Proteins
PubMed: 38782601
DOI: 10.26508/lsa.202402620 -
Biophysics Reports Apr 2024In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of...
In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.
PubMed: 38774352
DOI: 10.52601/bpr.2024.240008 -
Frontiers in Molecular Biosciences 2024The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological...
The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential. In polarized epithelial cells, GPI-APs are enriched at the apical surface, where they form small cholesterol-independent homoclusters and larger heteroclusters accommodating multiple GPI-AP species, all confined within areas of approximately 65-70 nm in diameter. Notably, GPI-AP homoclustering occurs in the Golgi apparatus through a cholesterol- and calcium-dependent mechanism that drives their apical sorting. Despite the critical role of Golgi GPI-AP clustering in their cell surface organization and the importance of cholesterol in heterocluster formation, the regulatory mechanisms governing GPI-AP surface organization, particularly in the context of epithelial polarity, remain elusive. Given that the actin cytoskeleton undergoes substantial remodeling during polarity establishment, this study explores whether the actin cytoskeleton regulates the spatiotemporal apical organization of GPI-APs in MDCK cells. Utilizing various imaging techniques (number and brightness, FRET/FLIM, and dSTORM coupled to pair correlation analysis), we demonstrate that the apical organization of GPI-APs, at different scales, does not rely on the actin cytoskeleton, unlike in fibroblastic cells. Interestingly, calcium chelation disrupts the organization of GPI-APs at the apical surface by impairing Golgi GPI-AP clustering, emphasizing the existence of an interplay among Golgi clustering, apical sorting, and surface organization in epithelial cells. In summary, our findings unveil distinct mechanisms regulating the organization of GPI-APs in cell types of different origins, plausibly allowing them to adapt to different external signals and different cellular environments in order to achieve specialized functions.
PubMed: 38774234
DOI: 10.3389/fmolb.2024.1360142 -
Journal of Cell Science May 2024Membrane trafficking, a fundamental cellular process encompassing the transport of molecules to specific organelles, endocytosis at the plasma membrane and protein... (Review)
Review
Membrane trafficking, a fundamental cellular process encompassing the transport of molecules to specific organelles, endocytosis at the plasma membrane and protein secretion, is crucial for cellular homeostasis and signalling. Cancer cells adapt membrane trafficking to enhance their survival and metabolism, and understanding these adaptations is vital for improving patient responses to therapy and identifying therapeutic targets. In this Review, we provide a concise overview of major membrane trafficking pathways and detail adaptations in these pathways, including COPII-dependent endoplasmic reticulum (ER)-to-Golgi vesicle trafficking, COPI-dependent retrograde Golgi-to-ER trafficking and endocytosis, that have been found in cancer. We explore how these adaptations confer growth advantages or resistance to cell death and conclude by discussing the potential for utilising this knowledge in developing new treatment strategies and overcoming drug resistance for cancer patients.
Topics: Humans; Neoplasms; Carcinogenesis; Animals; Cell Membrane; Endoplasmic Reticulum; Endocytosis; Protein Transport; Golgi Apparatus
PubMed: 38770683
DOI: 10.1242/jcs.260943 -
Communications Biology May 2024Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle...
Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle trafficking system, which includes Rab GTPases and their regulators, to assure the biogenesis and secretory of the organelles. Here we exploit the model apicomplexan organism Toxoplasma gondii that encodes a family of Rab GTPase Activating Proteins, TBC (Tre-2/Bub2/Cdc16) domain-containing proteins. Functional profiling of these proteins in tachyzoites reveals that TBC9 is the only essential regulator, which is localized to the endoplasmic reticulum (ER) in T. gondii strains. Detailed analyses demonstrate that TBC9 is required for normal distribution of proteins targeting to the ER, and the Golgi apparatus in the parasite, as well as for the normal formation of daughter inner membrane complexes (IMCs). Pull-down assays show a strong protein interaction between TBC9 and specific Rab GTPases (Rab11A, Rab11B, and Rab2), supporting the role of TBC9 in daughter IMC formation and early vesicular transport. Thus, this study identifies the only essential TBC domain-containing protein TBC9 that regulates early vesicular transport and IMC formation in T. gondii and potentially in closely related protists.
Topics: Toxoplasma; Protozoan Proteins; Endoplasmic Reticulum; rab GTP-Binding Proteins; GTPase-Activating Proteins; Golgi Apparatus; Protein Transport; Animals; Transport Vesicles
PubMed: 38762629
DOI: 10.1038/s42003-024-06310-6