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1.
J Cell Biol ; 223(10)2024 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-39007804

RESUMO

To breach the basement membrane, cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during Caenorhabditis elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives the expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid-producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the AC's invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, localizes to the ER and enriches in peroxisomes at the AC invasive front, and that the final transmembrane prenylation enzyme, ICMT-1, localizes to endoplasmic reticulum exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.


Assuntos
Membrana Basal , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Retículo Endoplasmático , Lipogênese , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Membrana Basal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Lipogênese/genética , Prenilação , Peroxissomos/metabolismo , Movimento Celular , Lisossomos/metabolismo
2.
Methods Enzymol ; 701: 175-236, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39025572

RESUMO

Biomembranes and vesicles cover a wide range of length scales. Indeed, small nanovesicles have a diameter of a few tens of nanometers whereas giant vesicles can have diameters up to hundreds of micrometers. The remodeling of giant vesicles on the micron scale can be observed by light microscopy and understood by the theory of curvature elasticity, which represents a top-down approach. The theory predicts the formation of multispherical shapes as recently observed experimentally. On the nanometer scale, much insight has been obtained via coarse-grained molecular dynamics simulations of nanovesicles, which provides a bottom-up approach based on the lipid numbers assembled in the two bilayer leaflets and the resulting leaflet tensions. The remodeling processes discussed here include the shape transformations of vesicles, their morphological responses to the adhesion of condensate droplets, the instabilities of lipid bilayers and nanovesicles, as well as the topological transformations of vesicles by membrane fission and fusion. The latter processes determine the complex topology of the endoplasmic reticulum.


Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Bicamadas Lipídicas/química , Membrana Celular/química , Membrana Celular/metabolismo , Fusão de Membrana/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Animais , Humanos
3.
Life Sci Alliance ; 7(9)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38981683

RESUMO

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.


Assuntos
Colágeno Tipo II , Retículo Endoplasmático , Pró-Colágeno , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Humanos , Pró-Colágeno/metabolismo , Colágeno Tipo II/metabolismo , Mutação , Células-Tronco Pluripotentes Induzidas/metabolismo , Cartilagem/metabolismo , Cartilagem/patologia , Dobramento de Proteína , Artrite/metabolismo , Artrite/genética , Osteoartrite/metabolismo , Osteoartrite/genética , Osteoartrite/patologia , Animais , Condrócitos/metabolismo
4.
Cells ; 13(13)2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38994992

RESUMO

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.


Assuntos
Retículo Endoplasmático , Resposta ao Choque Térmico , Termogênese , Humanos , Animais , Retículo Endoplasmático/metabolismo , Camundongos , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Hipertermia/metabolismo , Hipertermia/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo
5.
Int J Mol Sci ; 25(13)2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-39000521

RESUMO

The Na,K-ATPase is an α-ß heterodimer. It is well known that the Na,K-ATPase ß subunit is required for the biosynthesis and trafficking of the α subunit to the plasma membrane. During investigation of properties of human ATP1A3 mutations in 293 cells, we observed a reciprocal loss of endogenous ATP1A1 when expressing ATP1A3. Scattered reports going back as far as 1991 have shown that experimental expression of one subunit can result in reduction in another, suggesting that the total amount is strictly limited. It seems logical that either α or ß subunit should be rate-limiting for assembly and functional expression. Here, we present evidence that neither α nor ß may be limiting and that there is another level of control that limits the amount of Na,K-ATPase to physiological levels. We propose that α subunits compete for something specific, like a private chaperone, required to finalize their biosynthesis or to prevent their degradation in the endoplasmic reticulum.


Assuntos
Subunidades Proteicas , ATPase Trocadora de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Humanos , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Células HEK293 , Mutação , Animais , Retículo Endoplasmático/metabolismo
6.
Int J Mol Sci ; 25(13)2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-39000139

RESUMO

Epinephrine influences the function of pancreatic ß-cells, primarily through the α2A-adrenergic receptor (α2A-AR) on their plasma membrane. Previous studies indicate that epinephrine transiently suppresses insulin secretion, whereas prolonged exposure induces its compensatory secretion. Nonetheless, the impact of epinephrine-induced α2A-AR signaling on the survival and function of pancreatic ß-cells, particularly the impact of reprogramming after their removal from sustained epinephrine stimulation, remains elusive. In the present study, we applied MIN6, a murine insulinoma cell line, with 3 days of high concentration epinephrine incubation and 2 days of standard incubation, explored cell function and activity, and analyzed relevant regulatory pathways. The results showed that chronic epinephrine incubation led to the desensitization of α2A-AR and enhanced insulin secretion. An increased number of docked insulin granules and impaired Syntaxin-2 was found after chronic epinephrine exposure. Growth curve and cell cycle analyses showed the inhibition of cell proliferation. Transcriptome analysis showed the occurrence of endoplasmic reticulum stress (ER stress) and oxidative stress, such as the presence of BiP, CHOP, IRE1, ATF4, and XBP, affecting cellular endoplasmic reticulum function and survival, along with UCP2, OPA1, PINK, and PRKN, associated with mitochondrial dysfunction. Consequently, we conclude that chronic exposure to epinephrine induces α2A-AR desensitization and leads to ER and oxidative stress, impairing protein processing and mitochondrial function, leading to modified pancreatic ß-cell secretory function and cell fate.


Assuntos
Estresse do Retículo Endoplasmático , Epinefrina , Células Secretoras de Insulina , Insulina , Estresse Oxidativo , Animais , Epinefrina/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Camundongos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos alfa 2/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos
7.
Anal Chem ; 96(26): 10724-10731, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38952276

RESUMO

Carboxylesterase (CE), an enzyme widely present in organisms, is involved in various physiological and pathological processes. Changes in the levels of CEs in the liver may predict the presence of type 2 diabetes mellitus (T2DM). Here, a novel dicyanoisophorone (DCI)-based proximity-labeled far-red fluorescent probe DCI2F-Ac with endoplasmic reticulum targeting was proposed for real-time monitoring and imaging of the CEs activity. DCI2F-Ac featured very low cytotoxicity and biotoxicity and was highly selective and sensitive for CEs. Compared with traditional CEs probes, DCI2F-Ac was covalently anchored directly to CEs, thus effectively reducing the loss of in situ fluorescent signals due to diffusion. Through the "on-off" fluorescence signal readout, DCI2F-Ac was able to distinguish cell lines and screen for CEs inhibitors. In terms of endoplasmic reticulum (ER) stress, it was found that thapsigargin (Tg) induced upregulation of CEs levels but not tunicamycin (Tm), which was related to the calcium homeostasis of the ER. DCI2F-Ac could efficiently detect downregulated CEs in the livers of T2DM, and the therapeutic efficacy of metformin, acarbose, and a combination of these two drugs was assessed by tracking the fluctuation of CEs levels. The results showed that combining metformin and acarbose could restore CEs levels to near-normal levels with the best antidiabetic effect. Thus, the DCI2F-Ac probe provides a great opportunity to explore the untapped potential of CEs in liver metabolic disorders and drug efficacy assessment.


Assuntos
Carboxilesterase , Diabetes Mellitus Tipo 2 , Retículo Endoplasmático , Corantes Fluorescentes , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Carboxilesterase/metabolismo , Carboxilesterase/antagonistas & inibidores , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Animais , Camundongos , Imagem Óptica , Células Hep G2 , Estresse do Retículo Endoplasmático/efeitos dos fármacos
8.
Circ Res ; 135(2): 372-396, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38963864

RESUMO

Despite clinical and scientific advancements, heart failure is the major cause of morbidity and mortality worldwide. Both mitochondrial dysfunction and inflammation contribute to the development and progression of heart failure. Although inflammation is crucial to reparative healing following acute cardiomyocyte injury, chronic inflammation damages the heart, impairs function, and decreases cardiac output. Mitochondria, which comprise one third of cardiomyocyte volume, may prove a potential therapeutic target for heart failure. Known primarily for energy production, mitochondria are also involved in other processes including calcium homeostasis and the regulation of cellular apoptosis. Mitochondrial function is closely related to morphology, which alters through mitochondrial dynamics, thus ensuring that the energy needs of the cell are met. However, in heart failure, changes in substrate use lead to mitochondrial dysfunction and impaired myocyte function. This review discusses mitochondrial and cristae dynamics, including the role of the mitochondria contact site and cristae organizing system complex in mitochondrial ultrastructure changes. Additionally, this review covers the role of mitochondria-endoplasmic reticulum contact sites, mitochondrial communication via nanotunnels, and altered metabolite production during heart failure. We highlight these often-neglected factors and promising clinical mitochondrial targets for heart failure.


Assuntos
Insuficiência Cardíaca , Mitocôndrias Cardíacas , Humanos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Animais , Dinâmica Mitocondrial , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Metabolismo Energético , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia
9.
FASEB J ; 38(13): e23737, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38953724

RESUMO

Meningiomas are the most common primary intracranial tumors and account for nearly 30% of all nervous system tumors. Approximately half of meningioma patients exhibit neurofibromin 2 (NF2) gene inactivation. Here, NF2 was shown to interact with the endoplasmic reticulum (ER) calcium (Ca2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in IOMM-Lee, a high-grade malignant meningioma cell line, and the F1 subdomain of NF2 plays a critical role in this interaction. Functional assays indicated that NF2 promotes the phosphorylation of IP3R (Ser 1756) and IP3R-mediated endoplasmic reticulum (ER) Ca2+ release by binding to IP3R1, which results in Ca2+-dependent apoptosis. Knockout of NF2 decreased Ca2+ release and promoted resistance to apoptosis, which was rescued by wild-type NF2 overexpression but not by F1 subdomain deletion truncation overexpression. The effects of NF2 defects on the development of tumors were further studied in mouse models. The decreased expression level of NF2 caused by NF2 gene knockout or mutation affects the activity of the IP3R channel, which reduces Ca2+-dependent apoptosis, thereby promoting the development of tumors. We elucidated the interaction patterns of NF2 and IP3R1, revealed the molecular mechanism through which NF2 regulates IP3R1-mediated Ca2+ release, and elucidated the new pathogenic mechanism of meningioma-related NF2 variants. Our study broadens the current understanding of the biological function of NF2 and provides ideas for drug screening of NF2-associated meningioma.


Assuntos
Apoptose , Sinalização do Cálcio , Cálcio , Receptores de Inositol 1,4,5-Trifosfato , Neoplasias Meníngeas , Meningioma , Animais , Humanos , Camundongos , Cálcio/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patologia , Neoplasias Meníngeas/genética , Meningioma/metabolismo , Meningioma/patologia , Meningioma/genética , Neurofibromina 2
10.
Nat Commun ; 15(1): 5732, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38977690

RESUMO

Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.


Assuntos
Domínios Proteicos , Proteína de Ligação a Elemento Regulador de Esterol 2 , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Humanos , Serina Endopeptidases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Retículo Endoplasmático/metabolismo , Microscopia Crioeletrônica , Complexo de Golgi/metabolismo , Pró-Proteína Convertases/metabolismo , Pró-Proteína Convertases/genética , Colesterol/metabolismo , Animais , Células HEK293 , Transdução de Sinais
11.
Mol Plant Pathol ; 25(7): e13494, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39003585

RESUMO

Very-long-chain fatty acids (VLCFAs) regulate biophysical properties of cell membranes to determine growth and development of eukaryotes, such as the pathogenesis of the rice blast fungus Magnaporthe oryzae. The fatty acid elongase Elo1 regulates pathogenesis of M. oryzae by modulating VLCFA biosynthesis. However, it remains unknown whether and how Elo1 associates with other factors to regulate VLCFA biosynthesis in fungal pathogens. Here, we identified Ifa38, Phs1 and Tsc13 as interacting proteins of Elo1 by proximity labelling in M. oryzae. Elo1 associated with Ifa38, Phs1 and Tsc13 on the endoplasmic reticulum (ER) membrane to control VLCFA biosynthesis. Targeted gene deletion mutants Δifa38, Δphs1 and Δtsc13 were all similarly impaired as Δelo1 in vegetative growth, conidial morphology, stress responses in ER, cell wall and membrane. These deletion mutants also displayed severe damage in cell membrane integrity and failed to organize the septin ring that is essential for penetration peg formation and pathogenicity. Our study demonstrates that M. oryzae employs a fatty acid elongase complex to regulate VLCFAs for maintaining or remodelling cell membrane structure, which is important for septin-mediated host penetration.


Assuntos
Membrana Celular , Elongases de Ácidos Graxos , Proteínas Fúngicas , Oryza , Doenças das Plantas , Membrana Celular/metabolismo , Elongases de Ácidos Graxos/metabolismo , Elongases de Ácidos Graxos/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Septinas/metabolismo , Septinas/genética , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Ascomicetos/patogenicidade , Ascomicetos/genética
12.
Cell Mol Life Sci ; 81(1): 302, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39008111

RESUMO

DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Retículo Endoplasmático , Glicoproteínas de Membrana , Receptores Imunológicos , Via Secretória , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Retículo Endoplasmático/metabolismo , Via Secretória/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células HEK293 , Transdução de Sinais , Fagocitose/genética , Macrófagos/metabolismo , Transporte Proteico , Ligação Proteica , Animais , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Membrana Celular/metabolismo
13.
Anal Chem ; 96(28): 11557-11565, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38959297

RESUMO

Mitochondria (MT) and the endoplasmic reticulum (ER) maintain lipid and calcium homeostasis through membrane contacts, particularly MT-ER contacts (MERCs), spanning distances from 10 to 50 nm. However, the variation of different distance ranges and the metabolic factors influencing this variation remain poorly understood. This study employed microfluidic chip-based super-resolution microscopy in conjunction with a Moore-Neighbor tracing-incorporated organelle proximity analysis algorithm. This approach enabled precise three-dimensional localization of single-fluorescence protein molecules within narrow and irregular membrane proximities. It achieved lateral localization precision of less than 20 nm, resulting in a minimum MERC distance of approximately 8 nm in spatial and mean distances across multiple threshold ranges. Additionally, we demonstrated that the MERC distance variation was correlated with MT size rather than ER width. The proportion of each distance range varied significantly after the stimuli. Free cholesterol showed a negative correlation with various distances, while distances of 10-30 nm were associated with glucose, glutamine, and pyruvic acid. Furthermore, the 30-40 nm range was influenced by citric acid. These results underscore the role of advanced subcellular organelle analysis in elucidating the single-molecule behavior and organelle morphology in single-cell studies.


Assuntos
Retículo Endoplasmático , Mitocôndrias , Análise de Célula Única , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Microscopia de Fluorescência/métodos , Células HeLa
14.
Nat Commun ; 15(1): 5804, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987268

RESUMO

Environmental and physiological situations can challenge the balance between protein synthesis and folding capacity of the endoplasmic reticulum (ER) and cause ER stress, a potentially lethal condition. The unfolded protein response (UPR) restores ER homeostasis or actuates programmed cell death (PCD) when ER stress is unresolved. The cell fate determination mechanisms of the UPR are not well understood, especially in plants. Here, we integrate genetics and ER stress profiling with natural variation and quantitative trait locus analysis of 350 natural accessions of the model species Arabidopsis thaliana. Our analyses implicate a single nucleotide polymorphism to the loss of function of the general PCD regulator BON-ASSOCIATED PROTEIN2 (BAP2) in UPR outcomes. We establish that ER stress-induced BAP2 expression is antagonistically regulated by the UPR master regulator, inositol-requiring enzyme 1 (IRE1), and that BAP2 controls adaptive UPR amplitude in ER stress and ignites pro-death mechanisms in conditions of UPR insufficiency.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica de Plantas , Resposta a Proteínas não Dobradas , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Resposta a Proteínas não Dobradas/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Apoptose/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Locos de Características Quantitativas , Polimorfismo de Nucleotídeo Único
15.
J Cell Biol ; 223(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38958655

RESUMO

Export of secretory cargoes from the endoplasmic reticulum (ER) requires COPII proteins, which were first identified for their ability to coat small vesicles that bud from the ER. Recent data indicate that COPII proteins can also organize into a collar at the necks of tubules, as well as phase-separate into liquid-like condensates. Thus, COPII assemblies seem to be tailored to accommodate variations in the size and quantities of cargo secreted.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Retículo Endoplasmático , Transporte Proteico , Proteínas de Transporte Vesicular , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Animais
16.
Mol Plant Pathol ; 25(7): e13491, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38961768

RESUMO

Root-knot nematodes (RKNs) are microscopic parasitic worms able to infest the roots of thousands of plant species, causing massive crop yield losses worldwide. They evade the plant's immune system and manipulate plant cell physiology and metabolism to transform a few root cells into giant cells, which serve as feeding sites for the nematode. RKN parasitism is facilitated by the secretion in planta of effector molecules, mostly proteins that hijack host cellular processes. We describe here a conserved RKN-specific effector, effector 12 (EFF12), that is synthesized exclusively in the oesophageal glands of the nematode, and we demonstrate its function in parasitism. In the plant, MiEFF12 localizes to the endoplasmic reticulum (ER). A combination of RNA-sequencing analysis and immunity-suppression bioassays revealed the contribution of MiEFF12 to the modulation of host immunity. Yeast two-hybrid, split luciferase and co-immunoprecipitation approaches identified an essential component of the ER quality control system, the Solanum lycopersicum plant bap-like (PBL), and basic leucine zipper 60 (BZIP60) proteins as host targets of MiEFF12. Finally, silencing the PBL genes in Nicotiana benthamiana decreased susceptibility to Meloidogyne incognita infection. Our results suggest that EFF12 manipulates PBL function to modify plant immune responses to allow parasitism.


Assuntos
Retículo Endoplasmático , Tylenchoidea , Animais , Retículo Endoplasmático/metabolismo , Tylenchoidea/fisiologia , Tylenchoidea/patogenicidade , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Imunidade Vegetal , Nicotiana/parasitologia , Nicotiana/imunologia , Nicotiana/genética , Solanum lycopersicum/parasitologia , Solanum lycopersicum/imunologia , Solanum lycopersicum/genética , Doenças das Plantas/parasitologia , Doenças das Plantas/imunologia , Raízes de Plantas/parasitologia , Raízes de Plantas/imunologia , Interações Hospedeiro-Parasita
17.
Cell Death Dis ; 15(7): 480, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38965233

RESUMO

Chemotherapy persists as the primary intervention for breast cancer, with chemoresistance posing the principal obstacle to successful treatment. Herein, we show that cartilage oligomeric matrix protein (COMP) expression leads to increased cancer cell survival and attenuated apoptosis under treatment with several chemotherapeutic drugs, anti-HER2 targeted treatment, and endocrine therapy in several breast cancer cell lines tested. The COMP-induced chemoresistance was independent of the breast cancer subtype. Extracellularly delivered recombinant COMP failed to rescue cells from apoptosis while endoplasmic reticulum (ER)-restricted COMP-KDEL conferred resistance to apoptosis, consistent with the localization of COMP in the ER, where it interacted with calpain. Calpain activation was reduced in COMP-expressing cells and maintained at a lower level of activation during treatment with epirubicin. Moreover, the downstream caspases of calpain, caspases -9, -7, and -3, exhibited significantly reduced activation in COMP-expressing cells under chemotherapy treatment. Chemotherapy, when combined with calpain activators, rendered the cells expressing COMP more chemosensitive. Also, the anti-apoptotic proteins phospho-Bcl2 and survivin were increased in COMP-expressing cells upon chemotherapy. Cells expressing a mutant COMP lacking thrombospondin repeats exhibited reduced chemoresistance compared to cells expressing full-length COMP. Evaluation of calcium levels in the ER, cytosol, and mitochondria revealed that COMP expression modulates intracellular calcium homeostasis. Furthermore, patients undergoing chemotherapy or endocrine therapy demonstrated significantly reduced overall survival time when tumors expressed high levels of COMP. This study identifies a novel role of COMP in chemoresistance and calpain inactivation in breast cancer, a discovery with potential implications for anti-cancer therapy.


Assuntos
Apoptose , Neoplasias da Mama , Calpaína , Proteína de Matriz Oligomérica de Cartilagem , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Proteína de Matriz Oligomérica de Cartilagem/genética , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Calpaína/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos
18.
J Cell Biol ; 223(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-38949658

RESUMO

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.


Assuntos
Retículo Endoplasmático , Gotículas Lipídicas , Mitocôndrias , Gotículas Lipídicas/metabolismo , Humanos , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Metabolismo dos Lipídeos , Células HeLa , Células HEK293 , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/genética
19.
Int J Mol Sci ; 25(13)2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-39000233

RESUMO

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.


Assuntos
Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Hepatócitos , Isomerases de Dissulfetos de Proteínas , Transdução de Sinais , Tunicamicina , Chaperona BiP do Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Hepatócitos/metabolismo , Animais , Tunicamicina/farmacologia , Retículo Endoplasmático/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Linhagem Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Tapsigargina/farmacologia , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Sobrevivência Celular/efeitos dos fármacos
20.
Nat Commun ; 15(1): 6008, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-39019917

RESUMO

The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/metabolismo , Galactolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ligação Proteica , Receptores de Esteroides/metabolismo , Receptores de Esteroides/genética , Metabolismo dos Lipídeos , Lipidômica
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