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. 2015 Feb;98(1):65-72.
doi: 10.1016/j.yexmp.2014.12.003. Epub 2014 Dec 16.

Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis

Alejandro S Mendoza  1 , Jacques Dorce  1 , Yue Peng  1 , Barbara A French  1 , Brittany Tillman  1 , Jun Li  1 , Samuel W French  1
Affiliations

Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis

Alejandro S Mendoza et al. Exp Mol Pathol. 2015 Feb.

Abstract

Efficient management of misfolded or aggregated proteins in ASH and NASH is crucial for continued hepatic viability. Cellular protein quality control systems play an important role in the pathogenesis and progression of ASH and NASH. In a recent study, elevated Mca1 expression counteracted aggregation and accumulation of misfolded proteins and extended the life span of the yeast Saccharomyces cerevisiae (Hill et al, 2014). Mca1 may also associate with Ssa1 and Hsp104 in disaggregation and fragmentation of aggregated proteins and their subsequent degradation through the ER-associated degradation (ERAD) pathway. If degradation is not available, protection of the cellular environment from a misfolded protein is accomplished by its sequestration into two distinct inclusion bodies (Kaganovich et al., 2008) called the JUNQ (JUxta Nuclear Quality control compartment) and the IPOD (Insoluble Protein Deposit). Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 all play important roles in protein quality control systems. This study aims to measure the expression of Mca1 and related chaperones involved in protein quality control in alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) compared with normal control liver biopsies. Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 expressions were measured in three to six formalin-fixed paraffin embedded ASH and NASH liver biopsies and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. Mca1, Hsp104, Ydj1 and p62 were significantly upregulated compared to control (p<0.05) in ASH specimens. Hsp40 and VCP/p97 were also uptrending in ASH. In NASH, the only significant difference was the increased expression of Hsp104 compared to control (p<0.05). Ssa1 levels were uptrending in both ASH and NASH specimens. The upregulation of Mca1, Hsp104, Ydj1 and p62 in ASH may be elicited as a response to the chronic exposure of the hepatocytes to the toxicity of alcohol. Recruitment of Mca1, Hsp104, Ydj1 and p62 may indicate that autophagy, the ERAD, JUNQ, and IPOD systems are active in ASH. Whereas in NASH, elevated Hsp104 and uptrending Ssa1 levels may indicate that autophagy and IPOD may be the only active protein quality control systems involved.

Keywords: Alcoholic steatohepatitis; ERAD; Hsp104; Hsp40; IPOD; JUNQ; Mca1; Nonalcoholic steatohepatitis; Protein quality control; Ssa1; VCP/p97; Ydj1; p62.

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Figures

Figure 1
Figure 1
Print screen or screen snip pictures of representative Mca1 measurements from (A) ASH, (B) NASH, and (C) normal control liver samples. The presence of Mca1 was detected by the green immunofluorescence on the liver biopsy picture. A yellow tracer line converted this fluorescence intensity into quantitative figures and represented it as a green line on a graph, shown below each picture. The level of fluorescence intensity is dependent on the amount of Mca1. (A) ASH showed more green fluorescence compared to both (B) NASH, and (C) normal control liver biopsies. More fluorescence corresponded to the increase presence of Mca1. (D) Means with SD of Mca1 in ASH, NASH, and normal control liver. Levels of Mca1 in ASH are significantly elevated (p<0.05) compared to the normal control liver. There is no significant difference in the levels of Mca1 in NASH versus normal control liver. Comparing ASH versus NASH, data showed that Mca1 is significantly elevated in ASH (p<0.01). (×566 magnification)
Figure 2
Figure 2
(A) ASH, (B) NASH and (C) normal control measurements of Hsp104 levels. The presence of Hsp104 was detected by the green immunofluorescence on the liver biopsies. ASH and NASH showed slightly more green fluorescence compared to normal control liver biopsies. More fluorescence corresponded to the increase presence of Hsp104. (D) Means with SD of Hsp104 in ASH, NASH, and normal control liver. Levels of Hsp104 were significantly elevated in ASH (p<0.01) and NASH (p<0.05) compared to normal control. However, ASH and NASH did not show a significant difference from each other in Hsp104 levels. (×566 magnification)
Figure 3
Figure 3
(A), (B), (C) are representative snip pictures of Hsp40 measurements from ASH, NASH, and normal control, respectively. The presence of Hsp40 was detected by the green immunofluorescence on the liver biopsy. (A) ASH showed a little more green fluorescence compared to both (B) NASH and (C) normal control liver biopsies. More fluorescence corresponded to the increase presence of Hsp40. (D) Means with SD of Hsp40 in ASH, NASH, and normal control liver. Levels of Hsp40 did not show significant difference between control and ASH, as well as, between control and NASH. However, comparing ASH versus NASH, Hsp40 was significantly elevated in ASH (p<0.05). (×566 magnification)
Figure 4
Figure 4
(A) ASH, (B) NASH and (C) normal control measurements of Ydj1 levels. The presence of Ydj1 was detected by the green immunofluorescence on the liver biopsies. (A) ASH showed brighter green fluorescence compared to (B) NASH and (C) normal control. More fluorescence corresponded to the increase levels of Ydj1. (D) Means with SD of Ydj1 in ASH, NASH, and normal control liver biopsies. Ydj1 was significantly elevated in ASH (p<0.05) compared to normal control liver biopsies. Compared to NASH, Ydj1 was also significantly elevated in ASH (p<0.01). There is no significant difference in the level of Ydj1 between NASH and normal control liver. (×566 magnification)
Figure 5
Figure 5
(A) ASH, (B) NASH and (C) normal control measurements of Ssa1 levels. The presence of Ssa1 was detected by the green immunofluorescence on the liver biopsies. ASH, NASH and normal control liver biopsies showed almost the same green fluorescence intensity. (D) Means with SD of Ssa1 in ASH, NASH, and normal control liver. Measurements of Ssa1 did not show significant difference between the 3 groups. (×566 magnification)
Figure 6
Figure 6
(A) ASH, (B) NASH and (C) normal control measurements of VCP/p97 levels. The presence of VCP/p97was detected by the green immunofluorescence on the liver snip picture. The level of fluorescence intensity was dependent on the amount of VCP/p97. (A) ASH showed slightly greener fluorescence compared to (B) NASH. More fluorescence corresponded to the elevated levels of VCP/p97. (D) Means with SD of VCP/p97 in ASH, NASH, and normal control liver. VCP/p97levels did not show significant difference between ASH and control, nor between NASH and control. However, comparing ASH versus NASH, VCP/p97is significantly elevated in ASH (p<0.01). (×566 magnification)
Figure 7
Figure 7
(A) ASH, (B) NASH and (C) normal control measurements of p62. The presence of p62 was detected by the green immunofluorescence on the liver biopsies. ASH showed more green fluorescence compared to NASH and normal control liver biopsies. More fluorescence corresponded to the increase levels of p62. (D) Means with SD of p62 in ASH, NASH, and normal control liver. Measurements of p62 were significantly elevated in ASH (p<0.01) compared to normal control. Also, compared to NASH, p62 levels were significantly elevated in ASH (p<0.01). There is no difference in p62 levels between NASH and normal control.
Figure 8
Figure 8
MDBs in ASH(A) and NASH(B). Pictures of the liver biopsies on tricolor filter highlighting the presence of MDBs in both ASH and NASH specimens. MBDs are aggregated proteins and appear as red or bright yellow hyperdense areas.
Figure 9
Figure 9
(A) Summary of protein quality control mechanisms in ASH. Levels of Mca1, Hsp104, Hsp40, Ydj1, VCP/p97 and p62 were found to be elevated in ASH. These are involved in all four protein quality control mechanisms discussed: autophagy, the ERAD, the JUNQ and the IPOD systems. (B) Summary of protein quality control mechanisms in NASH. Only Ssa1 and Hsp104, associated with autophagy and the IPOD system, respectively, were found to be elevated in NASH specimens. Our data suggests that different protein quality control mechanisms are utilized in ASH versus NASH.
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