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Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

Dul JL, Davis DP, Williamson EK, Stevens FJ, Argon Y - J. Cell Biol. (2001)

Bottom Line: Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70.Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation.These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its beta sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

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SMA is dislocated from the ER and accumulates in vimentin-ringed aggresomes. Cells transfected with LEN (A and C) or SMA (B and D–F) were incubated without (A–D and F) or with (E) 10 μg/ml ALLN overnight. Cells were processed for immunofluorescence with anti–human kappa antibody only (green, A, B, and F), or double labeled with the same antibody and anti–vimentin antibody (red, C–E). LEN shows reticular and Golgi staining typical of a secreted protein (A and C); SMA (B, D, and E) exhibits diffuse staining of the cytosol and nucleus, plus concentrated staining of a perinuclear aggresome. In LEN-transfected cells (C), vimentin is distributed throughout the cytoplasm; in SMA-transfected cells (D and E), the network of filaments collapses, concentrating around the aggresome. In SMA-transfected cells treated with nocodazole (F), the aggresomes are dispersed.
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Figure 4: SMA is dislocated from the ER and accumulates in vimentin-ringed aggresomes. Cells transfected with LEN (A and C) or SMA (B and D–F) were incubated without (A–D and F) or with (E) 10 μg/ml ALLN overnight. Cells were processed for immunofluorescence with anti–human kappa antibody only (green, A, B, and F), or double labeled with the same antibody and anti–vimentin antibody (red, C–E). LEN shows reticular and Golgi staining typical of a secreted protein (A and C); SMA (B, D, and E) exhibits diffuse staining of the cytosol and nucleus, plus concentrated staining of a perinuclear aggresome. In LEN-transfected cells (C), vimentin is distributed throughout the cytoplasm; in SMA-transfected cells (D and E), the network of filaments collapses, concentrating around the aggresome. In SMA-transfected cells treated with nocodazole (F), the aggresomes are dispersed.

Mentions: Commonly, mutations in secretory proteins prevent exit from the ER such that the proteins accumulate in that organelle (Hammond and Helenius 1995). We previously determined that the intracellular localization of SMA did not fit this pattern (Davis et al. 2000). Rather, as shown again in Fig. 4, it displayed features that were unique. First, there was some ER staining, but it was frequently obscured by diffuse cytoplasmic staining. Second, in most cells, anti–kappa staining was prevalent throughout the nucleus, but the nucleoli were negative. Third, roughly one third to one half of SMA-expressing cells exhibited concentrated perinuclear staining that at first appeared similar to the Golgi staining observed in LEN-expressing cells (Fig. 4A and Fig. B). However, we have determined, through a series of experiments described in detail below, that these structures were instead perinuclear inclusions. Only one such SMA-induced perinuclear inclusion body was observed in each cell, and the frequency of cells with these inclusions was increased substantially when proteasomal activity was inhibited (see Fig. 6 and Fig. 8).


Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

Dul JL, Davis DP, Williamson EK, Stevens FJ, Argon Y - J. Cell Biol. (2001)

SMA is dislocated from the ER and accumulates in vimentin-ringed aggresomes. Cells transfected with LEN (A and C) or SMA (B and D–F) were incubated without (A–D and F) or with (E) 10 μg/ml ALLN overnight. Cells were processed for immunofluorescence with anti–human kappa antibody only (green, A, B, and F), or double labeled with the same antibody and anti–vimentin antibody (red, C–E). LEN shows reticular and Golgi staining typical of a secreted protein (A and C); SMA (B, D, and E) exhibits diffuse staining of the cytosol and nucleus, plus concentrated staining of a perinuclear aggresome. In LEN-transfected cells (C), vimentin is distributed throughout the cytoplasm; in SMA-transfected cells (D and E), the network of filaments collapses, concentrating around the aggresome. In SMA-transfected cells treated with nocodazole (F), the aggresomes are dispersed.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195780&req=5

Figure 4: SMA is dislocated from the ER and accumulates in vimentin-ringed aggresomes. Cells transfected with LEN (A and C) or SMA (B and D–F) were incubated without (A–D and F) or with (E) 10 μg/ml ALLN overnight. Cells were processed for immunofluorescence with anti–human kappa antibody only (green, A, B, and F), or double labeled with the same antibody and anti–vimentin antibody (red, C–E). LEN shows reticular and Golgi staining typical of a secreted protein (A and C); SMA (B, D, and E) exhibits diffuse staining of the cytosol and nucleus, plus concentrated staining of a perinuclear aggresome. In LEN-transfected cells (C), vimentin is distributed throughout the cytoplasm; in SMA-transfected cells (D and E), the network of filaments collapses, concentrating around the aggresome. In SMA-transfected cells treated with nocodazole (F), the aggresomes are dispersed.
Mentions: Commonly, mutations in secretory proteins prevent exit from the ER such that the proteins accumulate in that organelle (Hammond and Helenius 1995). We previously determined that the intracellular localization of SMA did not fit this pattern (Davis et al. 2000). Rather, as shown again in Fig. 4, it displayed features that were unique. First, there was some ER staining, but it was frequently obscured by diffuse cytoplasmic staining. Second, in most cells, anti–kappa staining was prevalent throughout the nucleus, but the nucleoli were negative. Third, roughly one third to one half of SMA-expressing cells exhibited concentrated perinuclear staining that at first appeared similar to the Golgi staining observed in LEN-expressing cells (Fig. 4A and Fig. B). However, we have determined, through a series of experiments described in detail below, that these structures were instead perinuclear inclusions. Only one such SMA-induced perinuclear inclusion body was observed in each cell, and the frequency of cells with these inclusions was increased substantially when proteasomal activity was inhibited (see Fig. 6 and Fig. 8).

Bottom Line: Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70.Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation.These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its beta sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

Show MeSH
Related in: MedlinePlus