This Is AuburnElectronic Theses and Dissertations

Interaction between SQSTM1/p62 and HDAC6: Impacts on the Aggresome-autophagy pathway and Microtubule Dynamics




Yan, Jin

Type of Degree



Biological Sciences


Aggresome-autophagy is a cellular degradation pathway that responds to protein misfolding stress. This pathway is particularly important because it maintains cellular homeostasis whenever the ubiquitin-proteasome degradation pathway is impaired. Misfolded proteins processed by the aggresome-autophagy pathway are first accumulated at the microtubule organizing center (MTOC) through retrograde transport. Proteins are formed into aggresomes which are then targeted by autophagic machinery for lysosome-dependent degradation. Research in recent years has revealed that the class II histone deacetylase HDAC6 plays important roles in regulation of aggresome formation and autophagic degradation. This regulation is achieved by interplay between HDAC6 and its interacting proteins. Interestingly, our laboratory’s long-time interest SQSTM1/p62 has also been shown to be critical for aggregation of misfolded proteins and their autophagic degradation. Similar to HDAC6, p62 also has a C-terminal ubiquitin-binding domain. It has been documented that HDAC6 and p62 may work together in regulation of aggresome-autophagy pathway. In Chapter 1 of this dissertation, I review the molecular mechanisms involved in this regulation and the literature related to HDAC6 and p62. In Chapter II, I further review the structure and function of HDAC6. Based on the current literature regarding the roles of HDAC6 and p62 in the aggresome-autophagy pathway, I hypothesized that p62 may directly interact with HDAC6. I further hypothesized that any such interaction might alter HDAC6 activity as well as impact its regulation of the aggresome-autophagy pathway and, potentially, microtubule dynamics. In order to investigate these hypotheses, two objectives are undertaken: (1) I conducted research aimed at identifing the possible interaction between p62 and HDAC6. Under this objective, I examined the functional effect on HDAC6 activity and the aggresome-autophagy pathway caused by loss of this interaction in p62KO mouse embryonic fibroblasts (MEFs); (2) I examined a hypothesized association between p62 and microtubules. Included in this objective was research aimed at identifying potential microtubule dynamic properties caused by altered HDAC6 activity observed in p62KO MEFs. In Chapter III, I show, using both biochemical and immunofluorescence approaches, that p62 indeed interact with HDAC6. Using deletion mutants, I mapped the interaction site on p62 to a region between the ZZ domain and TRAF6 domains. Using a similar approach, I found that the DD2 region of HDAC6 was the most likely site for its interaction with p62. I discovered that lack of this interaction in p62KO MEFs led to increased HDAC6 activity and reduced acetylation of two important HDAC6 substrates, α-tubulin and cortactin. I further show that p62 is essential for assembly of cortactin-actin networks at aggresome-like aggregates containing HDAC6 which subsequently plays a critical role during autophagy degradation of aggresomes. Overall, these results indicate that a loss of this interaction may impair the aggresome-autophagy pathway. In Chapter IV, I show, using both biochemical and immunofluoresence approaches, that p62 associates with microtubules. Absence of p62 in MEFs results in microtubule stabilization. However, pharmacological inhibition of HDAC6 in these cells did not stabilize microtubules, but enhanced binding of HDAC6 to microtubules. I also show that lack of p62 in MEFs leads to increased rate of microtubule reassembly compared to wild-type conditions. Collectively, these results indicate that microtubules from p62KO MEFs have different dynamic properties compared to those in wild-type cells. I conclude that both p62 deficiency and elevation of HDAC6 activity may contribute to altered microtubule dynamics in p62KO MEFs. Future studies towards better understanding “how” p62 interacts with HDAC6 were discussed in Chapter V. First, it would be meaningful to identify the residues on HDAC6 responsible for this interaction and regulation. Second, it would be imperative to explore possible functional redundancy between p62 and HDAC6 in the aggresome-autophagy pathway since HDAC6 activity is elevated with absence of p62 in MEFs. Third, it would also be worthwhile to examine the possible model in which p62 and HDAC6 may work together to recruit protein cargoes for aggresome formation. Last, it would be interesting to investigate whether decreased acetylated tubulin in p62 null background results in impaired microtubule trafficking which can contribute to Alzheimer’s Disease-like phenotype.