Abbreviations: ATG, autophagy-related; Bafi, bafilomycin A 1 ; BSA, bovine serum albumin; C. elegans, Caenorhabditis elegans; CAL-COCO2, calcium binding and coiled-coil domain 2; DAPI, 4', 6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DPH, 1, 6-diphenyl-1, 3, 5-hexatriene; eV, empty vector; FEZ, fasciculation and elongation protein zeta; GABARAP, GABA(A) receptor-associated protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; MAP1LC3, microtubule-associated protein 1 light chain 3; NBR1, neighbor of BRCA1 gene 1; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; RABGAP, RAB GTPase activating protein; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TBC domain, TRE2-BUB2-CDC16 domain.Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.

Dysfunction of autophagy and disturbed protein homeostasis are linked to the pathogenesis of human neurodegenerative diseases and the modulation of autophagy as the protein clearance process has become one key pharmacological target. Due to the role of sigma-1 receptors (Sig-1R) in learning and memory, and the described pleiotropic neuroprotective effects in various experimental paradigms, Sig-1R activation is recognized as one potential approach for prevention and therapy of neurodegeneration and, interestingly, in amyotrophic lateral sclerosis associated with mutated Sig-1R, autophagy is disturbed. Here we analyzed the effects of tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73), a muscarinic receptor ligand and Sig-1R agonist, on autophagy and proteostasis. We describe, at the molecular level, for the first time, that pharmacological Sig-1R activation a) enhances the autophagic flux in human cells and in Caenorhabditis elegans and b) increases proteostasis capacity, ultimately ameliorating paralysis caused by protein aggregation in C. elegans. ANAVEX2-73 is already in clinical investigation for the treatment of Alzheimer’s disease, and the novel activities of this compound on autophagy and proteostasis described here may have consequences for the use and further development of the Sig-1R as a drug target in the future. Moreover, our study defines the Sig-1R as an upstream modulator of canonical autophagy, which may have further implications for various conditions with dysfunctional autophagy, besides neurodegeneration.

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance.

No abstract

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.