Dataset Information

Ubiquitination is involved in secondary growth, not initial formation of polyglutamine protein aggregates in C. elegans.

ABSTRACT:

Background

Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates.

Results

The Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates.

Conclusions

These data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.

SUBMITTER: Skibinski GA

PROVIDER: S-EPMC3368771 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Publications

Ubiquitination is involved in secondary growth, not initial formation of polyglutamine protein aggregates in C. elegans.

Skibinski Gregory A GA Boyd Lynn L

BMC cell biology 20120411

<h4>Background</h4>Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The spe ...[more]

PMID: 22494772

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Project description:Elimination of paternal mitochondria after fertilization occurs in many species using the process of selective autophagy. The mechanism for targeting paternal mitochondria, but not maternal mitochondria, for elimination in the early embryo is not well understood. The results in this paper suggest that there are at least two different mechanisms for targeting paternal mitochondria for elimination: the first involving ubiquitination and a second involving a mitochondrial associated autophagy receptor, fndc-1. Elimination of paternal mitochondria can be visualized in embryos of the nematode, C. elegans. Paternal mitochondria enter the zygote at fertilization. Initially, they are closely associated with another sperm organelle, the membraneous organelle (MO). The MOs become ubiquitinated within minutes after fertilization. Simultaneous RNAi knockdown of two ubiquitin conjugating enzymes, ubc-18 and ubc-16, reduces MO ubiquitination. Loss of function of ubc-18 alone leads to loss of K48-linked polyubiquitin chains and halts the recruitment of proteasome to MOs. Interestingly, knockdown of ubc-18 or ubc-16 or the combination does not reduce the localization of K63-linked ubiquitin chains to MOs suggesting that some ubiquitin structure other than K63 chains is responsible for recruiting the autophagy machinery to MOs. Double knockdown (ubc-18/ubc-16) inhibits the recruitment of the autophagy protein, LGG-1 (homolog of LC3/GABARAP), to paternal organelles and causes the persistence of paternal mitochondria into the two cell stage. If paternal mitochondria are not eliminated via this early process, they are eventually removed from the embryo in a process that depends on the mitophagy adaptor protein, fndc-1. Thus, there are two redundant, but temporally distinct mechanisms that target paternal mitochondria for elimination in C. elegans. In addition to the involvement of ubiquitination in the elimination of paternal mitochondria, two subunits of the proteasome, rpn-10 and rad-23, are required for elimination of paternal mitochondria. These subunits are known to function as ubiquitin receptors and knockdown of either inhibits the recruitment of proteasome to ubiquitinated MOs. Their knockdown does not affect the localization of LGG-1 to paternal structures indicating that the proteasome is not required for autophagy membrane recruitment but might be involved in autophagosome maturation or its fusion with the lysosome.

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Dataset Information

Ubiquitination is involved in secondary growth, not initial formation of polyglutamine protein aggregates in C. elegans.

Background

Results

Conclusions

Publications

Ubiquitination is involved in secondary growth, not initial formation of polyglutamine protein aggregates in C. elegans.

Similar Datasets

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