Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation.

Meghana Somlapura,Benjamin Gottschalk,Thomas Rülicke,Pooja Lahiri,Iris Kufferath,Helmut Denk,Kurt Zatloukal,Wolfgang F Graier,Daniela Pabst

doi:10.3390/ijms22126227

Abstract

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62− total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.

Highlights

The SQSTM1 gene encodes the ubiquitously expressed sequestosome-1/p62 (p62), a stress-inducible scaffold protein with a variety of cellular functions
Hepatocyte-associated p62 aggregates are found as Mallory–Denk bodies (MDBs) in steatohepatitis and as intracytoplasmic hyaline bodies (IHBs) in hepatocellular carcinomas and copper toxicosis, and in the brain as Lewy bodies in Parkinson’s disease, neurofibrillary tangles in Alzheimer’s disease, and huntingtin aggregates in Huntington’s disease [8,12,13,14,15]
In order to elucidate the possible functions of p62-H1 and p62-H2 in protein aggregation, regarding hepatocellular inclusions, we investigated their interaction with keratins 8 and 18; in addition, we compared p62-H2 with a SH2 deletion mutant (p62∆SH2)

Summary

Introduction

The SQSTM1 gene encodes the ubiquitously expressed sequestosome-1/p62 (p62), a stress-inducible scaffold protein with a variety of cellular functions. Hepatocyte-associated p62 aggregates are found as Mallory–Denk bodies (MDBs) in steatohepatitis and as intracytoplasmic hyaline bodies (IHBs) in hepatocellular carcinomas and copper toxicosis, and in the brain as Lewy bodies in Parkinson’s disease, neurofibrillary tangles in Alzheimer’s disease, and huntingtin aggregates in Huntington’s disease [8,12,13,14,15] These proteinaceous inclusions are rich in amyloid-like structures and are mainly composed of p62, ubiquitin, chaperones, proteasome subunits as well as core proteins characteristic of the inclusion type (e.g., keratin in MDBs, α-synuclein in Lewy bodies or neurofilaments and tau in neurofibrillary tangles) [8,16,17]

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