Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors, such as chloroquine or E64D plus pepstatin A. Furthermore, the amount of free GFP fragments depends on the concentrations of these inhibitors. Unsaturating concentrations of chloroquine or bafilomycin A1 increased the level of free GFP fragments while saturating concentrations did not. Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP. However, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed. Therefore, caution must be used when conducting the GFP-LC3 cleavage assay as a determinant of autophagic flux. In order to accurately assess autophagy, it is more appropriate to assess GFP-LC3 cleavage in the presence or absence of saturating or unsaturating concentrations of chloroquine or bafilomycin A1 together with other autophagy markers, such as levels of p62 and endogenous LC3-II.

Cells respond to inflammatory disease states by releasing exosomes containing highly specific protein and RNA cargos, but how inflammation alters cargo specificity and secretion of exosomes is unknown. We show that increases in exosome secretion induced by either viral infection or LPS/ATP exposure result from inflammasome activation and subsequent caspase-1–dependent cleavage of the trafficking adaptor protein RILP. This cleaved form of RILP promotes the movement of multivesicular bodies toward the cell periphery and induces selective exosomal miRNA cargo loading. We have identified a common short sequence motif present in miRNAs that are selectively loaded into exosomes after RILP cleavage. This motif binds the RNA binding protein FMR1 and directs miRNA loading into exosomes via interaction with components of the ESCRT (endosomal sorting complex required for transport) pathway. These results indicate that inflammasome-mediated RILP cleavage, and sequence-specific interactions between miRNAs and FMR1, play a significant role in exosome cargo loading and enhanced secretion during cellular inflammatory responses.

Optimal therapeutic strategies for hepatocellular carcinoma (HCC) patients are still challenging due to the high recurrence rate after surgical resection and chemotherapy resistance. Growing evidence shows that genetic and epigenetic alterations are involved in HCC progression and resistance to therapy, however the molecular mechanisms underlying resistance to therapy have not been fully understood. Expression of SIRT7 in 17 paired paraffin-embedded HCC tissues and adjacent nontumoral liver tissues was examined by immunohistochemistry and Western blot. The mRNA expression of SIRT7 in 20 paired frozen HCC tissues and adjacent nontumoral liver tissues was analyzed by quantitative RT-PCR. The biologic consequences of overexpression and knockdown of SIRT7 in HCC therapy sensitivity were studied in vitro and in vivo. Interaction between SIRT7 and p53 were studied in HCC cell lines. SIRT7 expression was frequently upregulated in clinical HCC samples, and its expression was highly associated with TACE-resistance and poor survival (P = 0.008.) Depletion of SIRT7 from multiple liver cancer cell lines significantly increased doxorubicin toxicity while overexpression of SIRT7 largely abolished doxorubicin induced apoptosis. At the molecular level, we observed that SIRT7 interacts with and induces deacetylation of p53 at lysines 320 and 373. Deacetylated p53 showed significantly less affinity for the NOXA promoter and its transcription. In mouse xenografts, SIRT7 suppression increased doxorubicin induced p53 activation, inhibited tumor growth and induced apoptosis. The newly identified SIRT7-p53-NOXA axis partially illustrates the molecular mechanism of HCC resistance to therapy and represents a novel potential therapeutic target for HCC treatment.

Background & Aims: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western Diet Alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by scRNA seq and targeted Kupffer cell (KC) ablation to understand the diversity and function of liver macrophages in ALD. Approach and Results: In the WDA liver, KCs and infiltrating monocytes/macrophages (IMs) each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor mediated endocytosis and lipid metabolism, but most were predicted to be non-inflammatory and antifibrotic with one minor KC cluster having a pro-inflammatory and extracellular matrix degradation gene signature. IM clusters, in contrast, were predicted to be pro-inflammatory and pro-fibrotic. In vivo diphtheria toxin based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK-7 and Igf2bp3. Gene set enrichment analysis of whole liver RNAseq from the KC ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. Conclusions: In this ALD model, KCs are anti-inflammatory and are critical for maintenance of hepatocyte differentiation. IMs are largely pro-inflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to treatment of liver failure and fibrosis in ALD. Export

Extracellular vesicles (EVs) such as exosomes are loaded with specific biomolecules in order to perform cell-to-cell communication. Understanding the mechanism of selective cargo loading is important to better understand the physiological and pathological function of EVs. Here we describe a novel target of the E3 ligase TRIM25 and show that inflammation-mediated EV loading of the RNA binding protein FMR1 and its associated microRNA, miR-155, is promoted by TRIM25-mediated K63-ubiquitination of FMR1. This ubiquitination promotes an interaction between FMR1 and the EV loading machinery via the cleavage of the trafficking adaptor protein RILP. These interactions are lost when TRIM25 is knocked down. Loss of TRIM25 also prevents the loading of both FMR1 and miR-155. These findings suggest that inflammation-mediated loading of FMR1 and its associated microRNAs into the EV are dependent on K63-ubiquitination by TRIM25 and provide novel insights and tools to manipulate EV biogenesis for therapeutic benefit.