The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.Impairment of the ubiquitin-proteasome system is one of major causes of ubiquitin-positive inclusions found in various neurodegenerative diseases (1). Recent studies have identified the involvement of another degradation system, the autophagy-lysosomal pathway, in the formation of ubiquitin-positive inclusions as exemplified by the observation that autophagy-deficient mice exhibit substantial accumulation of such inclusions in tissues (2). p62/SQSTM1/A170, a multidomain protein found in ubiquitin-positive inclusions, has been shown to bind intracellular signaling factors (3-5). Accumulating evidence indicates that p62 is a receptor for ubiquitinated proteins that are targeted to the autophagosome for lysosomal degradation. Specifically, it is involved in autophagic elimination of damaged mitochondria, midbody rings, peroxisomes, and microbes (6 -10).The importance of p62 in autophagic degradation of proteins and organelles has been demonstrated by studies using tissuespecific autophagy-deficient mice. Elimination of Atg5 or Atg7, an essential gene in the formation of the autophagosome, in mouse neurons and hepatocytes resulted in toxicity accompanied by accumulation of ubiquitin-positive inclusions in the cells. In contrast, knock-out of both Atg7 and p62 (Atg7 Ϫ/Ϫ / p62 Ϫ/Ϫ ) caused a dramatic reduction in the amount of inclusions in both types of cells (2, 11). A similar result was also reported in fruit flies (12). These observations indicate that p62 is critically involved in the development of ubiquitin-positive inclusions that should be degraded via autophagy. In addition to playing a role in the autophagy-lysosomal pathway, p62 its...

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.

Adenosine triphosphate (ATP) is an immensely well-studied metabolite serving multiple key biochemical roles as the major chemical energy currency in living systems, a building block of ribonucleic acids, and a phosphoryl group donor in kinase-mediated signaling. Intriguingly, ATP has been recently proposed to act as a hydrotrope that inhibits aggregation of amyloidogenic proteins; however, the underlying mechanism and the general physicochemical effect that coexistence with ATP exerts on proteins remain unclear. By combining NMR spectroscopy and MD simulations, here we observed weak but unambiguously measurable and concentration-dependent noncovalent interactions between ATP and various proteins. The interactions were most pronounced for an intrinsically disordered protein (α-synuclein) and for residues in flexible regions (e.g., loops or termini) of two representative folded proteins (ubiquitin and the dimeric ubiquitin-binding domain of p62). As shown by solution NMR, a consequence of the ATP–protein interaction was altered hydration of solvent-exposed residues in the protein. The observation that ATP interacted with all three proteins suggests that ATP is a general nonspecific binder of proteins. Several complementary biophysical methods further confirmed that, at physiological concentrations of ∼5–10 mM, ATP starts to form oligomeric states via magnesium-chelating and chelation-independent mechanisms, in agreement with previous studies. Although the observed ATP–protein interaction was relatively weak overall, the high ratio of ATP (monomeric free ATP, mono- and divalent ion-bound ATP, oligomeric and chelated ATP) to proteins in cells suggests that most proteins are likely to encounter transient interactions with ATP (and chemically similar metabolites) that confer metabolite-mediated protein surface protection.

Background:The autophagic receptor NBR1 is commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Results: Molecular recognition of ubiquitin and polyubiquitin by NBR1 is described. Conclusion:The ubiquitin-associated domain of NBR1 shows unexpectedly high affinity for monoubiquitin but lacks polyubiquitin linkage specificity. Significance: NBR1 may be highly efficient at forming intracellular inclusions with ubiquitylated proteins via non-linkagespecific association with ubiquitin.

Shear stress can induce structural deformation of proteins, which might result in aggregate formation. Rheo-NMR spectroscopy has the potential to monitor structural changes in proteins under shear stress at the atomic level; however, existing Rheo-NMR methodologies have insufficient sensitivity to probe protein structure and dynamics. Here we present a simple and versatile approach to Rheo-NMR, which maximizes sensitivity by using a spectrometer equipped with a cryogenic probe. As a result, the sensitivity of the instrument ranks highest among the Rheo-NMR spectrometers reported so far. We demonstrate that the newly developed Rheo-NMR instrument can acquire high-quality relaxation data for a protein under shear stress and can trace structural changes in a protein during fibril formation in real time. The described approach will facilitate rheological studies on protein structural deformation, thereby aiding a physical understanding of shear-induced amyloid fibril formation.

Ubiquitin is a common post-translational modifier and its conjugation is a key signal for proteolysis by the proteasome. Because the molecular mass of ubiquitin is larger than that of other modifiers such as phosphate, acetyl, or methyl groups, ubiquitylation not only influences biochemical signaling, but also may exert physical effects on its substrate proteins by increasing molecular volume and altering shape anisotropy. Here we show that ubiquitylation destabilizes the fold of two proteins, FKBP12 and FABP4, and that elongation of the conjugated ubiquitin chains further enhances this destabilization effect. Moreover, NMR relaxation analysis shows that ubiquitylation induces characteristic structural fluctuations in the backbone of both proteins. These results suggest that the ubiquitylation-driven structural fluctuations lead to fold destabilization of its substrate proteins. Thus, physical destabilization by ubiquitylation may facilitate protein degradation by the proteasome.

Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation.