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1.
J Biol Chem ; 300(1): 105504, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38036174

RESUMEN

The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de la Membrana , Fosfoproteínas , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfoproteínas/metabolismo
2.
J Cell Biol ; 223(10)2024 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-38967608

RESUMEN

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the ß-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of ß-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/ß-catenin signaling during development.


Asunto(s)
Proteína Axina , Peroxisomas , Ubiquitina-Proteína Ligasas , Vía de Señalización Wnt , Peroxisomas/metabolismo , Peroxisomas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Humanos , Proteína Axina/metabolismo , Proteína Axina/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , beta Catenina/metabolismo , beta Catenina/genética , Células HEK293 , Transporte de Proteínas , Sistemas CRISPR-Cas
3.
bioRxiv ; 2024 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-38352406

RESUMEN

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose that RNF146 and tankyrase regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased tankyrase and RNF146-dependent degradation of non-peroxisomal substrates, including the beta-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development.

4.
Mol Biol Cell ; 35(6): br12, 2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38656789

RESUMEN

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.


Asunto(s)
Factor de Transcripción Activador 6 , Ciclo Celular , Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Respuesta de Proteína Desplegada , eIF-2 Quinasa , eIF-2 Quinasa/metabolismo , Humanos , Ciclo Celular/fisiología , Retículo Endoplásmico/metabolismo , Fosforilación , Factor 2 Eucariótico de Iniciación/metabolismo , Factor de Transcripción Activador 6/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Endorribonucleasas/metabolismo , Animales , Células HeLa , Estrés del Retículo Endoplásmico/fisiología
5.
J Biol Eng ; 18(1): 30, 2024 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-38649904

RESUMEN

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

6.
Nat Biotechnol ; 41(10): 1398-1404, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-36849829

RESUMEN

We describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated editing efficiencies by up to fivefold in K562, HEK293T, U2OS, iPS and primary T cells. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia pathway.

7.
bioRxiv ; 2023 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-37986852

RESUMEN

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

8.
bioRxiv ; 2023 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-37745580

RESUMEN

The heterohexameric AAA-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N-terminally bound co-factors. Here we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, S. cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N terminal domains of PEX1, CDC48, or NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of co-factors and substrates with Pex1/Pex6 ATPase activity.

9.
Cells ; 11(13)2022 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-35805150

RESUMEN

The AAA-ATPases Pex1 and Pex6 are required for the formation and maintenance of peroxisomes, membrane-bound organelles that harbor enzymes for specialized metabolism. Together, Pex1 and Pex6 form a heterohexameric AAA-ATPase capable of unfolding substrate proteins via processive threading through a central pore. Here, we review the proposed roles for Pex1/Pex6 in peroxisome biogenesis and degradation, discussing how the unfolding of potential substrates contributes to peroxisome homeostasis. We also consider how advances in cryo-EM, computational structure prediction, and mechanisms of related ATPases are improving our understanding of how Pex1/Pex6 converts ATP hydrolysis into mechanical force. Since mutations in PEX1 and PEX6 cause the majority of known cases of peroxisome biogenesis disorders such as Zellweger syndrome, insights into Pex1/Pex6 structure and function are important for understanding peroxisomes in human health and disease.


Asunto(s)
Proteínas de la Membrana , Peroxisomas , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Trifosfatasas/metabolismo , Homeostasis , Humanos , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo
10.
Nat Commun ; 9(1): 135, 2018 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-29321502

RESUMEN

Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/enzimología , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Conformación Proteica , Saccharomyces cerevisiae
11.
J Mol Biol ; 427(6 Pt B): 1375-1388, 2015 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-25659908

RESUMEN

Pex1 and Pex6 are Type-2 AAA+ ATPases required for the de novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis disorders in humans. Here, we show that the ATP-dependent complex of Pex1 and Pex6 from Saccharomyces cerevisiae is a heterohexamer with alternating subunits. Within the Pex1/Pex6 complex, only the D2 ATPase ring hydrolyzes ATP, while nucleotide binding in the D1 ring promotes complex assembly. ATP hydrolysis by Pex1 is highly coordinated with that of Pex6. Furthermore, Pex15, the membrane anchor required for Pex1/Pex6 recruitment to peroxisomes, inhibits the ATP-hydrolysis activity of Pex1/Pex6.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Hidrólisis , Procesamiento de Imagen Asistido por Computador , Inmunoprecipitación , Proteínas de la Membrana/química , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Peroxisomas/metabolismo , Fosfoproteínas/química , Conformación Proteica , Multimerización de Proteína , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Homología de Secuencia de Aminoácido
12.
Cold Spring Harb Perspect Biol ; 5(3): a013169, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23388626

RESUMEN

Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1's sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review.


Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Endorribonucleasas/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Respuesta de Proteína Desplegada/fisiología , eIF-2 Quinasa/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Polimerizacion , Levaduras
13.
Cell Host Microbe ; 13(5): 558-569, 2013 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-23684307

RESUMEN

The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cytosolic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen.


Asunto(s)
Toxina del Cólera/inmunología , Toxina del Cólera/metabolismo , ARN Helicasas DEAD-box/inmunología , Endorribonucleasas/inmunología , Endorribonucleasas/metabolismo , Inmunidad Innata , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Humanos , Unión Proteica , Receptores Inmunológicos
15.
Science ; 333(6051): 1891-4, 2011 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-21852455

RESUMEN

The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein-folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and ribonuclease domains. It is unclear what feature of ER stress Ire1 detects. We found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in yeast cells and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR.


Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Respuesta de Proteína Desplegada , Sitios de Unión , Catepsina A/química , Catepsina A/metabolismo , Polarización de Fluorescencia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glutatión Transferasa/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico
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