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Atg8-family proteins - structural features and molecular interactions in autophagy and beyond
(2020)
Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.
The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
Analysis of knockout/knockin mice that express a mutant FasL lacking the intracellular domain
(2009)
Fas ligand (FasL; CD178; CD95L) is a type II transmembrane protein belonging to the tumour necrosis factor family; its binding to the Fas receptor (CD95; APO-1) triggers apoptosis in the receptor-bearing cell. Signalling through this pathway plays a pivotal role during the immune response and in immune system homeostasis. Similar to other TNF family members, the intracellular domain has been reported to transmit signals to the inside of the FasL-bearing cell (reverse signalling). Recently, we identified the proteases ADAM10 and SPPL2a as molecules important for the processing of FasL. Protease cleavage releases the intracellular domain, which then is able to translocate to the nucleus and to repress reporter gene activity. To study the physiological importance of FasL reverse signalling in vivo, we established knockout/knockin mice with a FasL deletion mutant that lacks the intracellular portion (FasLDeltaIntra). Co-culture experiments confirmed that the truncated FasL protein is still capable of inducing apoptosis in Fas-sensitive cells. Preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular FasL domain does not alter the intracellular FasL localization in activated T cells. We are currently investigating signalling and proliferative capacities of T cells derived from homozygous FasLDeltaIntra mice to validate a co-stimulatory role of FasL reverse signalling.
The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL- and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domain-containing adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL·PSTPIP·PTP-PEST complexes which may contribute to FasL reverse signaling.