Exportin 1 modulates life span by regulating nucleolar dynamics via the autophagy protein LGG-1/GABARAP.

Altered nucleolar and ribosomal dynamics are key hallmarks of aging, but their regulation remains unclear. Building on the knowledge that the conserved nuclear export receptor Exportin 1 (XPO-1/XPO1) modulates proteostasis and life span, we systematically analyzed the impact of nuclear export on protein metabolism. Using transcriptomic and subcellular proteomic analyses in nematodes, we demonstrate that XPO-1 modulates the nucleocytoplasmic distribution of key proteins involved in nucleolar dynamics and ribosome function, including fibrillarin (FIB-1/FBL) and RPL-11 (RPL11). Silencing xpo-1 led to marked reduction in global translation, which was accompanied by decreased nucleolar size and lower fibrillarin levels. A targeted screen of known proteostatic mediators revealed that the autophagy protein LGG-1/GABARAP modulates nucleolar size by regulating RPL-11 levels, linking specific protein degradation to ribosome metabolism. Together, our study reveals that nucleolar size and life span are regulated by LGG-1/GABARAP via ribosome protein surveillance.

A novel method for determining antibody-dependent cellular phagocytosis.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to Fc gamma receptors (FcγRs) on the surface of immune cells or complement factors, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of treatment. Another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in in vitro. Most assays measure the association of target cells and macrophages; however, co-localization can represent cell attachment rather than internalization. Here, we describe the development of a novel method to accurately measure ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, the ADCP activity of antibody therapeutics can be accurately quantitated via flow cytometry.

Development of a kinetic antibody-dependent cellular cytotoxicity assay.

Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism of action (MOA) of monoclonal antibody (mAb) therapeutics. Target cells opsonized with therapeutic antibody bind and activate FcγR-bearing immune effector cells, resulting in target cell lysis. A key step in mAb drug development is the characterization of ADCC activity for its potential to inform mAb efficacy and safety. A number of in vitro assays are commonly used to assess ADCC. Most are endpoint assays that measure a surrogate marker of cell lysis. Newer imaging technologies allow direct measurement of ADCC-mediated cell lysis over time. In this study, we detail the development and characterization of a kinetic ADCC assay applicable to multiple target and effector cell types. This kinetic assay shows comparable sensitivity to an endpoint fluorescence release ADCC assay, while offering the advantages of a simpler set up and shorter assay time. Our results demonstrate that kinetic ADCC activity is a valid alternative assay format for measuring in vitro ADCC of mAbs.

Nuclear Export Inhibition Enhances HLH-30/TFEB Activity, Autophagy, and Lifespan.

Transcriptional modulation of the process of autophagy involves the transcription factor HLH-30/TFEB. In order to systematically determine the regulatory network of HLH-30/TFEB, we performed a genome-wide RNAi screen in C. elegans and found that silencing the nuclear export protein XPO-1/XPO1 enhances autophagy by significantly enriching HLH-30 in the nucleus, which is accompanied by proteostatic benefits and improved longevity. Lifespan extension via xpo-1 silencing requires HLH-30 and autophagy, overlapping mechanistically with several established longevity models. Selective XPO1 inhibitors recapitulated the effect on autophagy and lifespan observed by silencing xpo-1 and protected ALS-afflicted flies from neurodegeneration. XPO1 inhibition in HeLa cells enhanced TFEB nuclear localization, autophagy, and lysosome biogenesis without affecting mTOR activity, revealing a conserved regulatory mechanism for HLH-30/TFEB. Altogether, our study demonstrates that altering the nuclear export of HLH-30/TFEB can regulate autophagy and establishes the rationale of targeting XPO1 to stimulate autophagy in order to prevent neurodegeneration.