Sabine Schipper-Krom - Academia.edu (original) (raw)

Papers by Sabine Schipper-Krom

Journal of huntington's disease, Apr 16, 2024

Frontiers in Molecular Biosciences, Aug 20, 2019

The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of... more The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.

Journal of Biological Chemistry, Jun 1, 2013

Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the c... more Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the chaperones DNAJB6 and DNAJB8. Results: DNAJB6 and DNAJB8 prevent the aggregation of pure polyQ peptides. Conclusion: The polyQ tract is sufficient for DNAJB6 and DNAJB8 to prevent aggregation. Significance: By interacting with polyQ fragments, DNAJB6 and DNAJB8 reduce polyQ protein aggregation and may be potential therapeutic targets in polyQ disorders.

Ophthalmic Research, Oct 27, 2017

in and curcumin induced changes in proteasome-mediated proteolytic activity characterized by incr... more in and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β 2 and β 5i /β 1 and reduced activity of β 5 /β 1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Conclusions: Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells.

Biochemistry Research International, 2012

Huntington's disease is a progressive neurodegenerative disease, caused by a polyglutamine expans... more Huntington's disease is a progressive neurodegenerative disease, caused by a polyglutamine expansion in the huntingtin protein. A prominent hallmark of the disease is the presence of intracellular aggregates initiated by N-terminal huntingtin fragments containing the polyglutamine repeat, which recruit components of the ubiquitin-proteasome system. While it is commonly thought that proteasomes are irreversibly sequestered into these aggregates leading to impairment of the ubiquitin-proteasome system, the data on proteasomal impairment in Huntington's disease is contradictory. In addition, it has been suggested that proteasomes are unable to actually cleave polyglutamine sequences in vitro, thereby releasing aggregation-prone polyglutamine peptides in cells. Here, we discuss how the proteasome is involved in the various stages of polyglutamine aggregation in Huntington's disease, and how alterations in activity may improve clearance of mutant huntingtin fragments.

Frontiers in Molecular Biosciences

Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the... more Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the N-terminus of the HTT gene. The CAG repeat expansion translates into a polyglutamine expansion in the mutant HTT (mHTT) protein, resulting in intracellular aggregation and neurotoxicity. Lowering the mHTT protein by reducing synthesis or improving degradation would delay or prevent the onset of HD, and the ubiquitin-proteasome system (UPS) could be an important pathway to clear the mHTT proteins prior to aggregation. The UPS is not impaired in HD, and proteasomes can degrade mHTT entirely when HTT is targeted for degradation. However, the mHTT protein is differently ubiquitinated when compared to wild-type HTT (wtHTT), suggesting that the polyQ expansion affects interaction with (de) ubiquitinating enzymes and subsequent targeting for degradation. The soluble mHTT protein is associated with several ubiquitin-modifying enzymes, and various ubiquitin-modifying enzymes have been identified...

PLOS ONE

Huntington’s disease is an autosomal dominant heritable disorder caused by an expanded CAG trinuc... more Huntington’s disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αβ disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αβ activated proteasomes in various in vitro models showed that PA28αβ improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αβ in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αβ is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation.

Ophthalmic research, 2018

Curcumin has multiple biological effects including the modulation of protein homeostasis by the u... more Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α7, β5, and β5i were assessed. Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H2O2-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased ac...

Frontiers in Molecular Biosciences, 2019

Intracellular protein synthesis, folding, and degradation are tightly controlled processes to ens... more Intracellular protein synthesis, folding, and degradation are tightly controlled processes to ensure proper protein homeostasis. The proteasome is responsible for the degradation of the majority of intracellular proteins, which are often targeted for degradation via polyubiquitination. However, the degradation rate of proteins is also affected by the capacity of proteasomes to recognize and degrade these substrate proteins. This capacity is regulated by a variety of proteasome modulations including (1) changes in complex composition, (2) post-translational modifications, and (3) altered transcription of proteasomal subunits and activators. Various diseases are linked to proteasome modulation and altered proteasome function. A better understanding of these modulations may offer new perspectives for therapeutic intervention. Here we present an overview of these three proteasome modulating mechanisms to give better insight into the diversity of proteasomes.

Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the c... more Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the chaperones DNAJB6 and DNAJB8 Results: DNAJB6 and 8 prevent the aggregation of pure polyQ peptides. Conclusion: The polyQ tract is sufficient for DNAJB6 and 8 to prevent aggregation. Significance: By interacting with polyQ fragments, DNAJB6 and DNAJB8 reduce polyQ protein aggregation and may be potential therapeutic targets in polyQ disorders.

Frontiers in Molecular Biosciences

The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of... more The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.

Frontiers in Molecular Biosciences

The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of... more The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.

J Cell Sci, 2009

Several neurodegenerative disorders, including Huntington&amp... more Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein. Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.

Autophagy, 2014

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degrada... more Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome....

Journal of Cell Science, 2013

Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire carg... more Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire cargo from early endosomes, phagosomes and autophagosome. Defects in lysosomal dynamics cause severe neurodegenerative and developmental diseases, such as Niemann-Pick type C disease and ARC syndrome, yet little is known about the regulation of late endosomal fusion in a mammalian system. Mammalian endosomes destined for fusion need to be transported over very long distances before they tether to initiate contact. Here, we describe that lysosomal tethering and transport are combined processes co-regulated by one multi-protein complex: RAB7-RILP-ORP1L. We show that RILP directly and concomitantly binds the tethering HOPS complex and the p150 Glued subunit of the dynein motor. ORP1L then functions as a cholesterol-sensing switch controlling RILP-HOPS-p150 Glued interactions. We show that RILP and ORP1L control Ebola virus infection, a process dependent on late endosomal fusion. By combining recruitment and regulation of both the dynein motor and HOPS complex into a single multiprotein complex, the RAB7-RILP-ORP1L complex efficiently couples and regulates the timing of microtubule minus-end transport and fusion, two major events in endosomal biology.

Journal of Biological Chemistry, 2013

Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. ... more Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. A large network of molecular chaperones ensures protein quality control. Here, we show that within the Hsp70, Hsp110, and Hsp40 (DNAJ) chaperone families, members of a subclass of the DNAJB family (particularly DNAJB6b and DNAJB8) are superior suppressors of aggregation and toxicity of diseaseassociated polyglutamine proteins. The antiaggregation activity is largely independent of the N-terminal Hsp70-interacting J-domain. Rather, a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential. The SSF-SST region is involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with histone deacetylases (HDAC4, HDAC6, SIRT2). Inhibiting HDAC4 reduced DNAJB8 function. DNAJB8 is (de)acetylated at two conserved C-terminal lysines that are not involved in substrate binding, but do play a role in suppressing protein aggregation. Combined, our data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation.

Journal of Biological Chemistry, 2013

Background: Huntington disease is caused by an expanded polyglutamine repeat within the protein h... more Background: Huntington disease is caused by an expanded polyglutamine repeat within the protein huntingtin. Results: Proteasomal degradation of mutant huntingtin fragments is devoid of polyglutamine peptides as partial cleavage products. Conclusion: Mammalian proteasomes are capable of entirely degrading expanded polyglutamine sequences. Significance: Accelerating the mutant huntingtin degradation by the proteasomal pathway obviates toxic species and represents a beneficial therapeutic strategy.

Investigative Ophthalmology & Visual Science, 2013

Citation: Ramos de Carvalho JE, Klaassen I, Vogels IMC, et al. Complement factor C3a alters prote... more Citation: Ramos de Carvalho JE, Klaassen I, Vogels IMC, et al. Complement factor C3a alters proteasome function in human RPE cells and in an animal model of age-related RPE degeneration. Invest Ophthalmol Vis Sci.

Journal of huntington's disease, Apr 16, 2024

Frontiers in Molecular Biosciences, Aug 20, 2019

The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of... more The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.

Journal of Biological Chemistry, Jun 1, 2013

Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the c... more Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the chaperones DNAJB6 and DNAJB8. Results: DNAJB6 and DNAJB8 prevent the aggregation of pure polyQ peptides. Conclusion: The polyQ tract is sufficient for DNAJB6 and DNAJB8 to prevent aggregation. Significance: By interacting with polyQ fragments, DNAJB6 and DNAJB8 reduce polyQ protein aggregation and may be potential therapeutic targets in polyQ disorders.

Ophthalmic Research, Oct 27, 2017

in and curcumin induced changes in proteasome-mediated proteolytic activity characterized by incr... more in and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β 2 and β 5i /β 1 and reduced activity of β 5 /β 1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Conclusions: Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells.

Biochemistry Research International, 2012

Huntington's disease is a progressive neurodegenerative disease, caused by a polyglutamine expans... more Huntington's disease is a progressive neurodegenerative disease, caused by a polyglutamine expansion in the huntingtin protein. A prominent hallmark of the disease is the presence of intracellular aggregates initiated by N-terminal huntingtin fragments containing the polyglutamine repeat, which recruit components of the ubiquitin-proteasome system. While it is commonly thought that proteasomes are irreversibly sequestered into these aggregates leading to impairment of the ubiquitin-proteasome system, the data on proteasomal impairment in Huntington's disease is contradictory. In addition, it has been suggested that proteasomes are unable to actually cleave polyglutamine sequences in vitro, thereby releasing aggregation-prone polyglutamine peptides in cells. Here, we discuss how the proteasome is involved in the various stages of polyglutamine aggregation in Huntington's disease, and how alterations in activity may improve clearance of mutant huntingtin fragments.

Frontiers in Molecular Biosciences

Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the... more Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the N-terminus of the HTT gene. The CAG repeat expansion translates into a polyglutamine expansion in the mutant HTT (mHTT) protein, resulting in intracellular aggregation and neurotoxicity. Lowering the mHTT protein by reducing synthesis or improving degradation would delay or prevent the onset of HD, and the ubiquitin-proteasome system (UPS) could be an important pathway to clear the mHTT proteins prior to aggregation. The UPS is not impaired in HD, and proteasomes can degrade mHTT entirely when HTT is targeted for degradation. However, the mHTT protein is differently ubiquitinated when compared to wild-type HTT (wtHTT), suggesting that the polyQ expansion affects interaction with (de) ubiquitinating enzymes and subsequent targeting for degradation. The soluble mHTT protein is associated with several ubiquitin-modifying enzymes, and various ubiquitin-modifying enzymes have been identified...

PLOS ONE

Huntington’s disease is an autosomal dominant heritable disorder caused by an expanded CAG trinuc... more Huntington’s disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αβ disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αβ activated proteasomes in various in vitro models showed that PA28αβ improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αβ in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αβ is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation.

Ophthalmic research, 2018

Curcumin has multiple biological effects including the modulation of protein homeostasis by the u... more Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α7, β5, and β5i were assessed. Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H2O2-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased ac...

Frontiers in Molecular Biosciences, 2019

Intracellular protein synthesis, folding, and degradation are tightly controlled processes to ens... more Intracellular protein synthesis, folding, and degradation are tightly controlled processes to ensure proper protein homeostasis. The proteasome is responsible for the degradation of the majority of intracellular proteins, which are often targeted for degradation via polyubiquitination. However, the degradation rate of proteins is also affected by the capacity of proteasomes to recognize and degrade these substrate proteins. This capacity is regulated by a variety of proteasome modulations including (1) changes in complex composition, (2) post-translational modifications, and (3) altered transcription of proteasomal subunits and activators. Various diseases are linked to proteasome modulation and altered proteasome function. A better understanding of these modulations may offer new perspectives for therapeutic intervention. Here we present an overview of these three proteasome modulating mechanisms to give better insight into the diversity of proteasomes.

Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the c... more Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the chaperones DNAJB6 and DNAJB8 Results: DNAJB6 and 8 prevent the aggregation of pure polyQ peptides. Conclusion: The polyQ tract is sufficient for DNAJB6 and 8 to prevent aggregation. Significance: By interacting with polyQ fragments, DNAJB6 and DNAJB8 reduce polyQ protein aggregation and may be potential therapeutic targets in polyQ disorders.

Frontiers in Molecular Biosciences

The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of... more The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.

Frontiers in Molecular Biosciences

The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of... more The proteasome is a multi-catalytic molecular machine that plays a key role in the degradation of many cytoplasmic and nuclear proteins. The proteasome is essential and proteasome malfunction is associated with various disease pathologies. Proteasome activity depends on its catalytic subunits which are interchangeable and also on the interaction with the associated regulatory cap complexes. Here, we describe and compare various methods that allow the study of proteasome function in living cells. Methods include the use of fluorescently tagged proteasome subunits and the use of activity-based proteasome probes. These probes can be used in both biochemical assays and in microscopy-based experiments. Together with tagged proteasomes, they can be used to study proteasome localization, dynamics, and activity.

J Cell Sci, 2009

Several neurodegenerative disorders, including Huntington&amp... more Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein. Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.

Autophagy, 2014

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degrada... more Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome....

Journal of Cell Science, 2013

Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire carg... more Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire cargo from early endosomes, phagosomes and autophagosome. Defects in lysosomal dynamics cause severe neurodegenerative and developmental diseases, such as Niemann-Pick type C disease and ARC syndrome, yet little is known about the regulation of late endosomal fusion in a mammalian system. Mammalian endosomes destined for fusion need to be transported over very long distances before they tether to initiate contact. Here, we describe that lysosomal tethering and transport are combined processes co-regulated by one multi-protein complex: RAB7-RILP-ORP1L. We show that RILP directly and concomitantly binds the tethering HOPS complex and the p150 Glued subunit of the dynein motor. ORP1L then functions as a cholesterol-sensing switch controlling RILP-HOPS-p150 Glued interactions. We show that RILP and ORP1L control Ebola virus infection, a process dependent on late endosomal fusion. By combining recruitment and regulation of both the dynein motor and HOPS complex into a single multiprotein complex, the RAB7-RILP-ORP1L complex efficiently couples and regulates the timing of microtubule minus-end transport and fusion, two major events in endosomal biology.

Journal of Biological Chemistry, 2013

Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. ... more Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. A large network of molecular chaperones ensures protein quality control. Here, we show that within the Hsp70, Hsp110, and Hsp40 (DNAJ) chaperone families, members of a subclass of the DNAJB family (particularly DNAJB6b and DNAJB8) are superior suppressors of aggregation and toxicity of diseaseassociated polyglutamine proteins. The antiaggregation activity is largely independent of the N-terminal Hsp70-interacting J-domain. Rather, a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential. The SSF-SST region is involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with histone deacetylases (HDAC4, HDAC6, SIRT2). Inhibiting HDAC4 reduced DNAJB8 function. DNAJB8 is (de)acetylated at two conserved C-terminal lysines that are not involved in substrate binding, but do play a role in suppressing protein aggregation. Combined, our data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation.

Journal of Biological Chemistry, 2013

Background: Huntington disease is caused by an expanded polyglutamine repeat within the protein h... more Background: Huntington disease is caused by an expanded polyglutamine repeat within the protein huntingtin. Results: Proteasomal degradation of mutant huntingtin fragments is devoid of polyglutamine peptides as partial cleavage products. Conclusion: Mammalian proteasomes are capable of entirely degrading expanded polyglutamine sequences. Significance: Accelerating the mutant huntingtin degradation by the proteasomal pathway obviates toxic species and represents a beneficial therapeutic strategy.

Investigative Ophthalmology & Visual Science, 2013

Citation: Ramos de Carvalho JE, Klaassen I, Vogels IMC, et al. Complement factor C3a alters prote... more Citation: Ramos de Carvalho JE, Klaassen I, Vogels IMC, et al. Complement factor C3a alters proteasome function in human RPE cells and in an animal model of age-related RPE degeneration. Invest Ophthalmol Vis Sci.