Stephen E. Kaiser

Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection

Inactivation of the essential autophagy gene Atg5 results in selective accumulation of aggregation-prone proteins independently of substrate ubiquitination.

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.

Indirect inhibition of 26S proteasome activity in a cellular model of Huntington’s disease

Rather than directly impairing 26S proteasomes, misfolded huntingtin may disrupt cellular proteostasis and lead to competition for limited 26S proteasome capacity.

Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating

Synaptic vesicle biogenesis involves the recycling of synaptic vesicle components by clathrin‐mediated endocytosis from the presynaptic membrane. stoned B, a protein encoded by the stoned locus in Drosophila melanogaster has been shown to regulate vesicle recycling by interacting with synaptotagmin. We report here the identification and characterization of a human homolog of stoned B (hStnB). Human stoned B is a brain‐specific protein which co‐enriches with other endocytic proteins such as AP‐2 in a crude synaptic vesicle fraction and at nerve terminals. A domain with homology to the medium chain of adaptor complexes binds directly to both AP‐2 and synaptotagmin and competes with AP‐2 for the same binding site within synaptotagmin. Finally we show that the μ2 homology domain of hStnB stimulates the uncoating of both clathrin and AP‐2 adaptors from clathrin‐coated vesicles. We hypothesize that hStnB regulates synaptic vesicle recycling by facilitating vesicle uncoating.

The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water-filled Cavities.

SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7Å resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

Autophagy inhibition engages Nrf2-p62 Ub-associated signaling

Genetic inactivation of autophagy in liver or brain leads to the appearance of ubiquitin- and p62-positive inclusions coincident with liver dysfunction and neurodegeneration, respectively. In our recent study we measured the abundance of polyubiquitin species in autophagy-deficient tissues and demonstrated that a specific polyubiquitin chain linkage is not the decisive autophagic substrate-targeting signal. Instead our data suggest that aggregation or oligomerization of a misfolded protein, in the absence of detectable polyubiquitin modification, is an important signal for autophagic degradation. We determined that the ubiquitin accumulation observed upon autophagy inhibition is caused by p62-mediated activation of Nrf2 resulting in global transcriptional changes to ubiquitin-associated genes. Thus, substrate polyubiquitination does not appear to be the major autophagy substrate-targeting signal and the primary role of p62 appears to be Nrf2 activation, not ubiquitin-dependent substrate degradation. Selective targeting of proteins to distinct subcellular machineries is fundamental to the regulation of cellular decisions between catabolism and anabolism. In particular, ubiquitin (Ub)-mediated targeting of proteins is essential in maintaining cellular homeostasis, and has been predominantly associated with nonlysosomal-mediated degradation. However, liver- and brain-specific autophagy knockout mice exhibit accumulation of Ub- and p62-positive inclusions suggesting that Ub-modification targets cargo for selective autophagic degradation. Subsequent reports have established the requirement of p62 oligomerization for the appearance of Ub-positive inclusions and suggest that p62 is a selective ‘autophagy adaptor’ for recognition and delivery of Ub-modified cargo to autophagosomes. Autophagy contributes to the detoxification of misfolded, aggregated proteins commonly associated with neurodegenerative disorders (Alzheimer disease, Parkinson disease, Huntington disease, and Lou Gehrigs disease [amyotrophic lateral sclerosis] and prion encephalophathies) and the presence of these misfolded proteins also correlates with the appearance of Ub- and p62-positive inclusions. Revealing how proteins are ‘marked’ for selective recognition by the autophagy machinery is essential. To determine whether specific polyubiquitin linkages target substrates for selective autophagic degradation we used Ub absolute quantification (AQUA) mass spectrometry to measure the amount of Ub that accumulates in both liver- and brain-specific autophagy knockout mouse models. We observed a global increase in all Ub isopeptide and non-isopeptide species and the results were similar between two different autophagy-deficient models in two separate tissues. Moreover the increased levels of Ub conjugates observed in samples of Atg7-null tissues were suppressed when this mutation was combined with deletions of p62 or Nrf2. These results are significant for two reasons. First, Nrf2 controls expression levels of detoxification enzymes by regulating genes that contain an antioxidant response element (ARE). If substrate polyubiquitination was the major autophagy targeting signal then we would not expect Ub accumulation in autophagy-deficient tissue to depend upon the presence of this transcription factor. Second, whereas p62 has been hypothesized to be an adaptor facilitating the autophagic degradation of ubiquitinated substrates, it has been previously shown that loss of p62 protects tissues from autophagy deficiency by preventing Nrf2 activation. Our observations are consistent with a role for p62 in controlling Nrf2—and not with a role as a simple Ub-dependent autophagy adaptor. Our findings suggest that accumulation of Ub during autophagy deficiency is a result of Nrf2 stress signaling downstream of the multifunctional scaffolding protein p62 rather than polyubiquitin functioning as the substrate targeting signal for selective autophagy. To identify Ub-related Nrf2 target genes that might explain the Ub dysregulation observed in autophagy-deficient tissues, we used bioinformatics, functional genomics and RT-PCR. Nrf2 (and its heterodimeric binding partner Maf) transcription factor-binding sites were found to be enriched among Ub-associated genes and the autophagy network. Within the autophagy network, there are 35 Ub-associated Nrf2 targets, and 22 are differentially affected in Atg7-/- mice compared to wild-type mice. Importantly, these changes are reversed in the Atg7-/Nrf2-double knockout mice confirming Nrf2-dependent regulation. Our overall interpretation of these data is that autophagy deficiency results in a stress response that activates Nrf2, globally affecting numerous Ub-related proteins. It is important to note that upregulation of ARE-containing genes has been reported for numerous neurodegenerative disorders. Understanding the mechanistic details of how Nrf2 binds ARE elements within Ub-associated genes, with what binding partner it binds, and Nrf2 nuclear/cytoplasmic trafficking in response to autophagy inhibition/p62 accumulation will reveal the physiological relevance of Nrf2-p62 Ub-associated signaling in neurodegenerative disorders. Since our results from mouse indicate that polyubiquitination is not the major autophagy substrate targeting signal, we used quantitative mass spectrometry and flow cytometry to measure Ub-modification of a selective autophagy substrate in an autophagy-regulatable stable cell line. Ub has been implicated to function as a signal in several forms of selective autophagy such as pexophagy, mitophagy and xenophagy. However, in these studies, the dependence of p62 and Ub on autophagic clearance was shown by immunofluorescence studies in which the absence of p62 results in the loss of substrate puncta formation or Ub colocalization with the substrate. Colocalization, though indicative of a signaling role, does not demonstrate covalent Ub modification of substrates. Our work addresses directly whether substrate modification by polyubiquitin chains targets proteins for selective autophagy and enables us to identify additional features of selective substrates. We developed a flow cytometry cell-based assay to measure selective autophagy using an autophagy-regulatable cell line stably expressing a bicistronic reporter construct containing both the misfolded, aggregation-prone protein huntingtin with an expanded polyglutamine tract (htt(Q47)) fused to GFP (green fluorescent protein), and the non-aggregation-prone protein cherry chFP (cherry fluorescent protein). Following autophagy shut-off, the reporter cell lines provide the ability to quantify and compare the relative accumulation of the two different fluorescent reporters, where autophagic selectivity is indicated by a ratio of greater than one. Using our flow cytometry assay we measured the selective accumulation of the aggregation-prone protein htt(Q47) compared to the non-aggregation prone protein chFP and although there is global accumulation of polyubiquitin chains following autophagy shut-off there is no increase in polyubiquitin-modified htt(Q47). Overall, these data demonstrate that aggregation or oligomerization of a misfolded protein, in the absence of detectable Ub-modification, results in selective accumulation following genetic ablation of autophagy. Based on these observations the major conclusion of our manuscript is that oligomerization targets proteins for selective autophagy in mammalian cells, and that polyubiquitination does not appear to be the major autophagy targeting signal; the primary role of p62 in autophagy deficiency appears to be Nrf2 activation, not Ub-dependent substrate degradation. Moreover, our data also demonstrate the broad importance of Nrf2-driven Ub signaling as an important cellular detoxification mechanism acting in addition to the arsenal of Nrf2-oxidative stress genes, and suggest that sustained activation could be detrimental to the cell (Fig. 1). Figure 1 Schematic of Ub-associated signaling regulated by the Nrf2-p62 axis. The Keap1-Cul3-Rbx1 E3 Ub ligase maintains low levels of Nrf2 in the cell. In response to oxidative insult, accumulation of misfolded, aggregation-prone proteins or additional unknown ...

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.

Structures of Atg7-Atg3 and Atg7-Atg10 reveal noncanonical mechanisms of E2 recruitment by the autophagy E1

Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.

Binding to E1 and E3 is mutually exclusive for the human autophagy E2 Atg3

Ubiquitin‐like proteins (UBLs) are activated, transferred and conjugated by E1‐E2‐E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3‐mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12∼Atg5‐Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3‐binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.

Ubiquitin-like modifiers

UBLs (ubiquitin-like proteins) are a major class of eukaryotic post-translational modifiers. UBLs are attached to numerous cellular proteins and to other macromolecules, thereby regulating a wide array of cellular processes. In this chapter we highlight a subset of UBLs and describe their regulatory roles in the cell.