Ravi Manjithaya

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Duran et al. in this issue).

PpAtg30 tags peroxisomes for turnover by selective autophagy.

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

Molecular mechanism and physiological role of pexophagy

Pexophagy is a selective autophagy process wherein damaged and/or superfluous peroxisomes undergo vacuolar degradation. In methylotropic yeasts, where pexophagy has been studied most extensively, this process occurs by either micro‐ or macropexophagy: processes analogous to micro‐ and macroautophagy. Recent studies have identified specific factors and illustrated mechanisms involved in pexophagy. Although mechanistically pexophagy relies heavily on the core autophagic machinery, the latest findings about the role of auxiliary pexophagy factors have highlighted specialized membrane structures required for micropexophagy, and shown how cargo selectivity is achieved and how cargo size dictates the requirement for these factors during pexophagy. These insights and additional observations in the literature provide a framework for an understanding of the physiological role(s) of pexophagy.

A yeast MAPK cascade regulates pexophagy but not other autophagy pathways

The S. cerevisiae Slt2p MAPK cascade picks out peroxisomes for autophagy-mediated degradation (pexophagy) but is not involved in turnover of other cellular components.

Roles of Pichia pastoris Uvrag in vacuolar protein sorting and the phosphatidylinositol 3-kinase complex in phagophore elongation in autophagy pathways.

Although it has been established that Atg6/Beclin 1, the phosphatidylinositol 3-kinase (PI3K) Vps34, and associated proteins have direct or indirect roles in autophagic pathways in both mammals and yeasts, the elucidation of these roles and the proteins required for them is ongoing. The involvement of the Beclin 1-binding protein, UVRAG, has been a particular source of disagreement. We found that PpAtg6 is required for all autophagic pathways that have been identified in the yeast Pichia pastoris, as well as for the carboxypeptidase Y (PpCPY) vacuolar protein sorting pathway. We localized PpAtg6 to the phagophore assembly site (PAS) and observed its continued presence at that site as the isolation membrane grew from it and matured into a pexophagosome. PpUvrag, however, was required for proper PpCPY sorting, but not for any autophagic pathway. Rather, the defects in all autophagic pathways observed when PpUvrag was overexpressed support its presence in a complex that competes with the PI3K complex required for autophagy.

Rationally Designed Peptidomimetic Modulators of Aβ Toxicity in Alzheimer's Disease

Alzheimers disease is one of the devastating illnesses mankind is facing in the 21st century. The main pathogenic event in Alzheimers disease is believed to be the aggregation of the β-amyloid (Aβ) peptides into toxic aggregates. Molecules that interfere with this process may act as therapeutic agents for the treatment of the disease. Use of recognition unit based peptidomimetics as inhibitors are a promising approach, as they exhibit greater protease stability compared to natural peptides. Here, we present peptidomimetic inhibitors of Aβ aggregation designed based on the KLVFF (P1) sequence that is known to bind Aβ aggregates. We improved inhibition efficiency of P1 by introducing multiple hydrogen bond donor-acceptor moieties (thymine/barbiturate) at the N-terminal (P2 and P3), and blood serum stability by modifying the backbone by incorporating sarcosine (N-methylglycine) units at alternate positions (P4 and P5). The peptidomimetics showed moderate to good activity in both inhibition and dissolution of Aβ aggregates as depicted by thioflavin assay, circular dichroism (CD) measurements and microscopy (TEM). The activity of P4 and P5 were studied in a yeast cell model showing Aβ toxicity. P4 and P5 could rescue yeast cells from Aβ toxicity and Aβ aggregates were cleared by the process of autophagy.

Dual role of autophagy in hallmarks of cancer

Evolutionarily conserved across eukaryotic cells, macroautophagy (herein autophagy) is an intracellular catabolic degradative process targeting damaged and superfluous cellular proteins, organelles, and other cytoplasmic components. Mechanistically, it involves formation of double-membrane vesicles called autophagosomes that capture cytosolic cargo and deliver it to lysosomes, wherein the breakdown products are eventually recycled back to the cytoplasm. Dysregulation of autophagy often results in various disease manifestations, including neurodegeneration, microbial infections, and cancer. In the case of cancer, extensive attention has been devoted to understanding the paradoxical roles of autophagy in tumor suppression and tumor promotion. In this review, while we summarize how this self-eating process is implicated at various stages of tumorigenesis, most importantly, we address the link between autophagy and hallmarks of cancer. This would eventually provide a better understanding of tumor dependence on autophagy. We also discuss how therapeutics targeting autophagy can counter various transformations involved in tumorigenesis. Finally, this review will provide a novel insight into the mutational landscapes of autophagy-related genes in several human cancers, using genetic information collected from an array of cancers.

Role of autophagy in unconventional protein secretion.

In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

ABSTRACT Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.

Septins are involved at the early stages of macroautophagy in S. cerevisiae

ABSTRACT Autophagy is a conserved cellular degradation pathway wherein double-membrane vesicles called autophagosomes capture long-lived proteins, and damaged or superfluous organelles, and deliver them to the lysosome for degradation. Septins are conserved GTP-binding proteins involved in many cellular processes, including phagocytosis and the autophagy of intracellular bacteria, but no role in general autophagy was known. In budding yeast, septins polymerize into ring-shaped arrays of filaments required for cytokinesis. In an unbiased genetic screen and in subsequent targeted analysis, we found autophagy defects in septin mutants. Upon autophagy induction, pre-assembled septin complexes relocalized to the pre-autophagosomal structure (PAS) where they formed non-canonical septin rings at PAS. Septins also colocalized with autophagosomes, where they physically interacted with the autophagy proteins Atg8 and Atg9. When autophagosome degradation was blocked in septin-mutant cells, fewer autophagic structures accumulated, and an autophagy mutant defective in early stages of autophagosome biogenesis (atg1Δ), displayed decreased septin localization to the PAS. Our findings support a role for septins in the early stages of budding yeast autophagy, during autophagosome formation. This article has an associated First Person interview with the first author of the paper. Highlighted Article: Septin proteins, which form cytoskeletal filaments and bind membranes, are required for efficient assembly of functional autophagic structures in budding yeast cells.