Lamin aggregation is an early sensor of porphyria-induced liver injury

Abstract

SummaryOxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or Nmethyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress

“…For example, as the current findings and earlier work (11) show, lamins are more susceptible to porphyrin-mediated protein aggregation as compared with keratins. In contrast, in the dark, keratins appear to be more susceptible than lamins (Figs.…”

“…Lamin Aggregation Parallels the Increase in PP-IX Accumulation-We tested lamin aggregation after exogenous PP-IX treatment and ALAϩDFO stimulation, given our prior finding that lamins are a sensitive marker for PP-IX-induced liver damage because of their exquisite sensitivity to aggregate in association with porphyria-associated liver injury (11). Indeed, dramatic lamin B1 and A/C aggregation was detected in both Hepa and HepG2 cells (Fig.…”

“…Antibodies were obtained from Santa Cruz Biotechnology Inc., Dallas, TX (lamin A/C, protein disulfide isomerase (PDI), and ubiquitin (Ub)); Abcam, Cambridge, MA (lamin B1); Cell Sig-naling, Boston, MA (TSPO, CHOP, BiP, calnexin, IRE1␣, and PERK); and Thermo Scientific (keratin 8 (clone TS1) and keratin 18 (clone DC10)). Mass spectrometry was carried out as described (11). The mass spectrometric data were sorted according to three criteria: (a) proteins with molecular mass less than 260 kDa, (b) proteins detected in both light-harvested and dark-harvested samples, and (c) Ͼ5-fold change in peptideto-spectrum matches (as compared with untreated control).…”

“…Recently, we demonstrated that the nuclear intermediate filament protein, lamin, aggregates in response to PP-IX-mediated liver damage (11). Profound aggregation of lamins A/C and B1 was observed in two different mouse models of protoporphyria and liver injury: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a Fech mutation, which leads to PP-IX accumulation.…”

Upregulated heme biosynthesis, an exploitable vulnerability in MYCN-driven leukemogenesis

“…We extended this to demonstrate that specific inhibition of ferrochelatase, which mediates the terminal step of heme biosynthesis with the selective inhibitor, N-methyl protoporphyrin (20), produced comparable suppression of basal, maximal, and spare respiratory capacity (Figure 2, H and I). Of note, suppression of heme synthesis was associated with an apparent compensatory increase in glycolysis, which was incapable of promoting self-renewal (Figure 2, F and J).…”