Improving the performance of mutation-based evolving artificial neural networks with self-adaptive mutations.

Neuroevolution is a promising approach for designing artificial neural networks using an evolutionary algorithm. Unlike recent trending methods that rely on gradient-based algorithms, neuroevolution can simultaneously evolve the topology and weights of neural networks. In neuroevolution with topological evolution, handling crossover is challenging because of the competing conventions problem. Mutation-based evolving artificial neural network is an alternative topology and weights neuroevolution approach that omits crossover and uses only mutations for genetic variation. This study enhances the performance of mutation-based evolving artificial neural network in two ways. First, the mutation step size controlling the magnitude of the parameter perturbation is automatically adjusted by a self-adaptive mutation mechanism, enabling a balance between exploration and exploitation during the evolution process. Second, the structural mutation probabilities are automatically adjusted depending on the network size, preventing excessive expansion of the topology. The proposed methods are compared with conventional neuroevolution algorithms using locomotion tasks provided in the OpenAI Gym benchmarks. The results demonstrate that the proposed methods with the self-adaptive mutation mechanism can achieve better performance. In addition, the adjustment of structural mutation probabilities can mitigate topological bloat while maintaining performance.

Cyclization of ubiquitin chains reinforces their recognition by ZNF216.

Lys48-linked ubiquitin chains, regulating proteasomal protein degradation, are known to include cyclized forms. This cyclization hinders recognition by many downstream proteins by occluding the Ile44-centered patch. In contrast, the A20-like Znf domain of ZNF216 (a ubiquitin-binding protein, A20 Znf) is expected to bind to cyclic ubiquitin chains via constitutively solvent-exposed surfaces. However, the underlying interaction mechanism remains unclear. Here, our ITC and NMR experiments collectively showed that cyclization did not interfere with and even slightly enhance the molecular recognition of diubiquitin by A20 Znf. This effect is explained by the cyclization-induced repression of conformational dynamics in diubiquitin and an enlarged molecular interface in the complex. Thus, these results suggest that cyclic ubiquitin chains can be involved in regulation of ZNF216-dependent proteasomal protein degradation.

Isolation of viruses, including mollivirus, with the potential to infect Acanthamoeba from a Japanese warm temperate zone.

Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.

Conformational Fluctuations and Induced Orientation of a Protein, Its Solvation Shell, and Bulk Water in Weak Non-Unfolding External Electric Fields.

Extreme external electric fields have been reported to disrupt the tertiary structure of stably folded proteins; however, the effects of weaker electric fields on many biomolecules, especially net-uncharged proteins, and on the surrounding aqueous environment have been rarely discussed. To explore these effects at the atomic level, here, we have used molecular dynamics simulations to estimate rotational motion and induced structural fluctuations in the model protein ubiquitin and its hydration layer due to applied non-unfolding electrostatic fields. When exposed to weak electric fields of up to 0.2 V nm-1, ubiquitin displayed competition between internal structure-maintaining molecular interactions and the external orienting force, which disrupted the local structure in certain regions of the protein. Moreover, relative to hydration water, bulk water showed a greater tendency to align with the electric field, indicating that the presence of protein caused hydration water to acquire rotational mobility different from that in a pure-water system. The differential influence of the applied electric field on the hydration and bulk water surrounding ubiquitin will be common to almost all (nonmembrane) biomacromolecules. Our findings highlight the importance of local dipoles and their electric polarizability even in net-uncharged biomolecules.

Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity.

Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain-interacting protein), specifically synthesizes "head-to-tail" poly-Ub chains, which are linked via the N-terminal methionine α-amino and C-terminal carboxylate of adjacent Ub units and are thus commonly called "linear" poly-Ub chains. Linear ubiquitin chain assembly complex-assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ub via its Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub-bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain's role in full-length HOIL-1L.

An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi-domain protein.

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub2 ). As expected, cyclization increased the thermal stability of Ub2 and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub2 . Furthermore, cyclization repressed the interdomain motion in Ub2 , but cyclic Ub2 still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub2 to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub2 .

Diversity in Infection Specificity between the Bloom-forming Microalga Heterosigma akashiwo and Its dsDNA Virus, Heterosigma akashiwo Virus.

Heterosigma akashiwo virus (HaV) is a dsDNA virus that infects the bloom-forming raphidoflagellate Heterosigma akashiwo. Both the host and its virus are phenotypically diverse in terms of infection specificity. Their relationships have been examined based on the occurrence or absence of algal lysis following virus inoculation; however, variations in the strain-level host-virus relationship regarding infectivity and lysis rates remain unclear. Therefore, we performed a series of cross-infectivity tests using 60 H. akashiwo and 22 HaV strains isolated from the coastal waters of western Japan. The host strains were divided into 5 different groups and viruses into 4 groups. Using a representative strain from each group, algal lysis was observed in 14 of the (5×4=) 20 host-virus combinations; the concentration of infectious units in each HaV suspension was then assessed using the most probable number (MPN) assay on the five host strains. Virus titers ranged between 1.1×101 and 2.1×107 infectious units mL-1; the titer of each viral lysate was differently estimated using distinct H. akashiwo strains as hosts. These results suggest that (1) a clonal viral lysate comprises virions with different intraspecific infection specificities and/or (2) the efficiency and error rates of each intracellular replication process vary in each host-virus combination.

Transcriptome analysis of Aurantiochytrium limacinum under low salt conditions.

Aurantiochytrium limacinum can accumulate high amounts of omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). Although salinity affects the DHA content, its impact on the metabolic pathway responsible for DHA production in A. limacinum is not completely understood. To address this issue, we investigated the transcriptional profile of A. limacinum under hypoosmotic stress. We first cultured A. limacinum under typical and low salinity for RNA sequencing, respectively. Transcriptome analyses revealed that 933 genes exhibited significant changes in expression under hypoosmotic conditions, of which 81.4% were downregulated. Strikingly, A. limacinum downregulated genes related to polyketide synthesis and fatty acid synthase pathways, while upregulating ß-oxidation-related genes. In accordance with this, DHA production significantly decreased under hypoosmotic conditions, while antioxidant-related genes were significantly upregulated. Considering that ß-oxidation of fatty acids generates energy and reactive oxygen species (ROS), our results suggest that A. limacinum utilizes fatty acids for energy to survive under hypoosmotic conditions and detoxifies ROS using antioxidant systems.

Ecological Dynamics of Broad- and Narrow-Host-Range Viruses Infecting the Bloom-Forming Toxic Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa is predicted to interact and coexist with diverse broad- and narrow-host-range viruses within a bloom; however, little is known about their affects on Microcystis population dynamics. Here, we developed a real-time PCR assay for the quantification of these viruses that have different host ranges. During the sampling period, total Microcystis abundance showed two peaks in May and August with a temporary decrease in June. The Microcystis population is largely divided into three phylotypes based on internal transcribed sequences (ITS; ITS types I to III). ITS I was the dominant phylotype (66% to 88%) except in June. Although the ITS II and III phylotypes were mostly less abundant, these phylotypes temporarily increased to approximately equivalent abundances of the ITS I population in June. During the same sampling period, the abundances of the broad-host-range virus MVGF_NODE331 increased from April to May and from July to October with a temporary decrease in June, in which its dynamics were in proportion to the increase of total Microcystis abundances regardless of changes in host ITS population composition. In contrast, the narrow-host-range viruses MVG_NODE620 and Ma-LMM01 were considerably less abundant than the broad-host-range virus and generally did not fluctuate in the environment. Considering that M. aeruginosa could increase the abundance and sustain the bloom under the prevalence of the broad-host-range virus, host abundant and diverse antiviral mechanisms might contribute to coexistence with its viruses. IMPORTANCE The bloom-forming toxic cyanobacterium Microcystis aeruginosa interacts with diverse broad- and narrow-host-range viruses. However, the dynamics of the Microcystis population (at the intraspecies level) and viruses with different host ranges remain unknown. Our real-time PCR assays unveiled that the broad-host-range virus gradually increased in abundance over the sampling period, in proportion to the increase in total Microcystis abundance regardless of changes in genotypic composition. The narrow-host-range viruses were considerably less abundant than the broad-host-range virus and did not generally fluctuate in the environment. The expansion and maintenance of the Microcystis bloom even under the increased infection by the broad-host-range virus suggested that highly abundant and diverse antiviral mechanisms allowed them to coexist with viruses under selective pressure. This paper expands our knowledge about the ecological dynamics of Microcystis viruses and provides potential insights into their coexistence with their host.

Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene.

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by ß-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5 days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from ß-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

Rheo-NMR Spectroscopy for Cryogenic-Probe-Equipped NMR Instruments to Monitor Protein Aggregation.

Cryogenic-probe-based Rheo-NMR spectroscopy is a recently developed methodology to obtain solution NMR spectra of protein samples in situ under external shear. It is applicable to atomic-resolution monitoring of protein aggregation in situ, thereby aiding understanding of the transient structural changes and state conversion of amyloidogenic proteins, which are strongly associated with the both the onset and the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Here, we present detailed experimental procedures for the instrumental setup and practical tips for preparation of NMR measurement to analyze protein aggregation by this technique. This protocol will thus aid future Rheo-NMR spectroscopic studies not only of protein aggregation but also of other phenomena related to shear stress, such as shear-induced viscosity increase and shear-enhanced crystallization. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Setup of a Rheo-NMR Instrument Basic Protocol 2: Adjustment of the Vertical and Horizontal Positions of the Glass Stick Basic Protocol 3: Monitoring Protein Aggregation by Rheo-NMR Spectroscopy.

Transcriptional responses of Aurantiochytrium limacinum under light conditions.

AIMS: Astaxanthin-producing protist Aurantiochytrium limacinum can accumulate higher amounts of astaxanthin under light conditions; however, little is known about the impact of light exposure on its metabolism. Here, we investigated the transcriptional profile of A. limacinum under light conditions. METHODS AND RESULTS: Transcriptomic analyses revealed that 962 genes of A. limacinum showed a significant change in expression under light conditions, most of which (94.5%) were downregulated. Furthermore, gene ontology enrichment analysis indicated that A. limacinum mainly downregulated genes associated with cell motility, proliferation and gene expression processes, whose activities depend on ATP as an energy source. Additionally, the quantification of carotenoid and its transcripts suggested that ß-carotene and astaxanthin biosynthesis pathways were rate-limiting and tightly regulated steps, respectively. In comparison, these processes were enhanced under light conditions. CONCLUSIONS: Considering that astaxanthin accumulation was highly correlated with reactive oxygen species (ROS) levels in microalgae, our results suggest that A. limacinum reduces ATP consumption to decrease the occurrence of ROS in mitochondria while accumulating astaxanthin to prevent ROS damage. SIGNIFICANCE AND IMPACT OF STUDY: This study provides novel insights into the impact of light exposure on A. limacinum metabolism, thereby facilitating a complete understanding of this protist for efficient astaxanthin production.

Structural Insights into Methylated DNA Recognition by the Methyl-CpG Binding Domain of MBD6 from Arabidopsis thaliana.

Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells.

Rigorous analysis of the interaction between proteins and low water-solubility drugs by qNMR-aided NMR titration experiments.

Drugs are designed and validated based on physicochemical data on their interactions with target proteins. For low water-solubility drugs, however, quantitative analysis is practically impossible without accurate estimation of precipitation. Here we combined quantitative NMR with NMR titration experiments to rigorously quantify the interaction of the low water-solubility drug pimecrolimus with its target protein FKBP12. Notably, the dissociation constants estimated with and without consideration of precipitation differed by more than tenfold. Moreover, the method enabled us to quantitate the FKBP12-pimecrolimus interaction even under a crowded condition established using the protein crowder BSA. Notably, the FKBP12-pimecrolimus interaction was slightly hampered under the crowded environment, which is explained by transient association of BSA with the drug molecules. Collectively, the described method will contribute to both quantifying the binding properties of low water-solubility drugs and to elucidating the drug behavior in complex crowded solutions including living cells.

Effects of Weak Nonspecific Interactions with ATP on Proteins.

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.

Expression, solubility monitoring, and purification of the co-folded LUBAC LTM domain by structure-guided tandem folding in autoinducing cultures.

The linear ubiquitin chain assembly complex tethering motif (LUBAC-LTM) domain is composed of two different accessory LUBAC components (HOIL-1L and SHARPIN) but folds as a single globular domain. Targeted disruption of the intricate LTM-LTM interaction destabilizes LUBAC in lymphoma cells, thereby attenuating LUBAC stability, which highlights that targeting the interaction between the two LTM motifs is a promising strategy for the development of new agents against cancers that depend on LUBAC activity for their survival. To further screen for small-molecule inhibitors that can selectively disrupt the LTM-LTM interaction, it is necessary to obtain high-purity samples of the LTM domain. Ideally, such a sample would not contain any components other than the LTM itself, so that false positives (molecules binding to other parts of LUBAC) could be eliminated from the screening process. Here we report a simple strategy that enabled successful bacterial production of the isolated LUBAC LTM domain in high yield and at high purity. The strategy combines (1) structural analysis highlighting the possibility of tandem expression in the SHARPINL™ to HOIL-1LL™ direction; (2) bacterial expression downstream of EGFP to efficiently monitor expression and solubility; (3) gentle low-temperature folding using autoinduction. Formation of stably folded LTM was verified by size-exclusion chromatography and heteronuclear NMR spectroscopy. From 200-ml cultures sufficient quantities (~7 mg) of high-purity protein for structural studies could be obtained. The presented strategy will be beneficial for LUBAC LTM-based drug-screening efforts and likely serve as a useful primer for similar cases, i.e., whenever a smaller folded fragment is to be isolated from a larger protein complex for site-specific downstream applications.

Multiple-State Monitoring of SOD1 Amyloid Formation at Single-Residue Resolution by Rheo-NMR Spectroscopy.

Formation of protein aggregates or fibrils entails the conversion of soluble native protein monomers via multiple molecular states. No spectroscopic techniques have succeeded in capturing the transient molecular-scale events of fibrillation in situ. Here we report residue- and state-specific real-time monitoring of the fibrillation of amyotrophic lateral sclerosis-related SOD1 by rheology NMR (Rheo-NMR) spectroscopy. Under moderately denaturing conditions, where NMR signals of folded and unfolded monomeric SOD1 are simultaneously observable, the cross-peak intensities of folded monomeric SOD1 decreased faster than those of the unfolded species, and a 310-helix in folded SOD1 was deformed prior to global unfolding. Furthermore, real-time protein dynamics analysis identified residues involved in the core structure formation of SOD1 oligomers. Our findings provide insight into local and global unfolding events in SOD1 and fibril formation. This Rheo-NMR analysis will be applicable not only to atomic-level monitoring of other amyloidogenic proteins but also to quantification of shear-induced structural changes of non-amyloidogenic proteins and elucidation of shear-enhanced chemical phenomena such as viscosity increase and crystallization of various solution-state compounds.

Backbone resonance assignments of the A2 domain of mouse von Willebrand factor.

von Willebrand factor (vWF) is an adhesive plasma protein that is important for platelet adhesion in normal hemostasis in response to vascular injury. Although large vWF multimers are released from storage granules of platelets and (sub-)endothelial cells in response to hemostatic stimuli, for normal physiological function, vWF multimers are required to be cleaved into smaller multimeric forms. The plasma metalloproteinase ADAMTS13 specifically cleaves the peptide bond located in the middle of the A2 domain of vWF (vWF-A2), but the cleavage site is buried inside the structure of vWF and is difficult to access in the absence of elevated flow shear stress. On the other hand, in the presence of high vascular shear stress, the structure of vWF-A2 is supposed to be unfolded, thereby becoming accessible for proteolysis by ADAMTS13. However, the atomic-level mechanism underlying shear-induced structural changes of vWF-A2 remains unclear and to date no solution NMR information is available. In this study, we present the backbone 1H, 13C, and 15N resonance assignments of mouse vWF-A2; side chain assignments of 13Cß are also provided. Secondary structure propensity analysis based on the assigned chemical shifts showed that mouse vWF-A2 forms similar secondary structures in solution to the previously determined crystal structure of human vWF-A2. The obtained NMR assignment data will contribute to an atomic-level characterization of shear-induced unfolding of vWF-A2 in solution.

Molecular recognition and deubiquitination of cyclic K48-linked ubiquitin chains by OTUB1.

Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains.