All roads lead to the lysosome: exploring the degradation of TNIP1 by selective autophagy

Selective autophagy is important for maintaining cellular homeostasis. Generally, autophagy is considered cytoprotective and anti-inflammatory, acting to limit infection and accumulation of deleterious material. Key to this function is the ability to select cargo to be degraded, and here, selective autophagy receptors play a central role. In this thesis, we show that the anti-inflammatory and pro-survival adaptor TNIP1 is a selective autophagy substrate. Moreover, we identify two LIR motifs in TNIP1, of which LIR2 is primarily responsible for binding to ATG8 proteins. While TNIP1 is constitutively degraded by autophagy in resting cells, inflammatory signaling via TLR3 resulted in increased degradation of TNIP1. Activation of the kinase TBK1 was demonstrated to directly phosphorylate LIR2 in TNIP1, leading to enhanced ATG8 interaction and increased TNIP1 degradation by ATG7-dependent macroautophagy. The degradation of TNIP1 correlated with the increased activation of downstream inflammatory signaling. This suggests that the reduction of TNIP1 protein levels by autophagy upon inflammatory stimuli occurs to allow the mounting of a robust inflammatory response. Many studies of TNIP1 function have been done using mouse models. We found that human LIR1 is impaired by the presence of a proline, making LIR2 the main functional LIR in human TNIP1. In mice, however, LIR1 can augment binding to LC3A in conjunction with LIR2. Nonetheless, we discover that the constitutive turnover of human and mouse TNIP1 occurs independently of LIRs, contrary to the inflammation-induced degradation by macroautophagy. We show that a part of TNIP1 that binds to TAX1BP1 and NBR1 is required for lysosomal degradation. Our study of the constitutive turnover of TNIP1 highlights the existence of alternative routes to the lysosome beyond canonical macroautophagy. Finally, we provide an overview of the ancestral selective autophagy receptor NBR1. Here, we explore the evolution of NBR1 and selective autophagy, and discuss the role of NBR1 in different forms of selective autophagy

NBR1: The archetypal selective autophagy receptor

NBR1 was discovered as an autophagy receptor not long after the first described vertebrate autophagy receptor p62/SQSTM1. Since then, p62 has currently been mentioned in >10,000 papers on PubMed, while NBR1 is mentioned in <350 papers. Nonetheless, evolutionary analysis reveals that NBR1, and likely also selective autophagy, was present already in the last eukaryotic common ancestor (LECA), while p62 appears first in the early Metazoan lineage. Furthermore, yeast-selective autophagy receptors Atg19 and Atg34 represent NBR1 homologs. NBR1 is the main autophagy receptor in plants that do not contain p62, while most animal taxa contain both NBR1 and p62. Mechanistic studies are starting to shed light on the collaboration between mammalian NBR1 and p62 in the autophagic degradation of protein aggregates (aggrephagy). Several domains of NBR1 are involved in cargo recognition, and the list of known substrates for NBR1-mediated selective autophagy is increasing. Lastly, roles of NBR1 in human diseases such as proteinopathies and cancer are emerging

The inflammation repressor TNIP1/ABIN-1 is degraded by autophagy following TBK1 phosphorylation of its LIR

The inflammatory repressor TNIP1/ABIN-1 is important for keeping in check inflammatory and cell-death pathways to avoid potentially dangerous sustained activation of these pathways. We have now found that TNIP1 is rapidly degraded by selective macroautophagy/autophagy early (0–4 h) after activation of TLR3 by poly(I:C)-treatment to allow expression of pro-inflammatory genes and proteins. A few hours later (6 h), TNIP1 levels rise again to counteract sustained inflammatory signaling. TBK1-mediated phosphorylation of a TNIP1 LIR motif regulates selective autophagy of TNIP1 by stimulating interaction with Atg8-family proteins. This is a novel level of regulation of TNIP1, whose protein level is crucial for controlling inflammatory signaling

TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0–4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response

Regulation of gap junction intercellular communication by connexin ubiquitination: physiological and pathophysiological implications

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junctions have a wide diversity of physiological functions, playing critical roles in both excitable and non-excitable tissues. Gap junction channels are formed by integral membrane proteins called connexins. Inherited or acquired alterations in connexins are associated with numerous diseases, including heart failure, neuropathologies, deafness, skin disorders, cataracts and cancer. Gap junctions are highly dynamic structures and by modulating the turnover rate of connexins, cells can rapidly alter the number of gap junction channels at the plasma membrane in response to extracellular or intracellular cues. Increasing evidence suggests that ubiquitination has important roles in the regulation of endoplasmic reticulum-associated degradation of connexins as well as in the modulation of gap junction endocytosis and post-endocytic sorting of connexins to lysosomes. In recent years, researchers have also started to provide insights into the physiological roles of connexin ubiquitination in specific tissue types. This review provides an overview of the advances made in understanding the roles of connexin ubiquitination in the regulation of gap junction intercellular communication and discusses the emerging physiological and pathophysiological implications of these processes