Thermodynamic Characterization of LC3/GABARAP:Ligand Interactions by Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.

High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.

CoZr nanocomposites in a ceramic-metal AlOx(OH)y/Al matrix with a different Co/Zr ratio and its potential for syngas processing.

We investigated the possibility of synthesizing Co nanoparticles in CoZrnH/AlOx(OH)y/Al ceramic-metal catalysts and controlling the catalytic properties of these nanoparticles in syngas conversion by changing the Co/Zr ratio. The CoZr nanocomposites were obtained from metal powders by mechanochemical activation in a high-energy mill under an argon atmosphere, followed by treatment with hydrogen at high pressure and room temperature. Ceramic-metal catalysts were prepared by mixing the corresponding CoZrnH powder nanocomposite (30 wt%) with powdered aluminum (70 wt%), hydrothermal treatment of the mixture and subsequent calcination. The materials were characterized with a set of physicochemical methods: powder X-ray diffraction, scanning electron microscopy, 59Co internal field nuclear magnetic resonance spectroscopy, and temperature programmed reduction. Catalytic studies were performed in a laboratory fixed-bed flow reactor at 2 MPA and 210-270 °C. It is shown that the activity in syngas conversion to C5+ hydrocarbons and selectivity to methane and C2-C4 hydrocarbons depend on the Co/Zr ratio. Thus, with an increase in the zirconium content in the samples, the interaction of metal cobalt with metal zirconium improves in the process of mechanical activation and subsequent treatment with hydrogen. The destruction of the agglomerates of crystallites of metallic cobalt in the form of ß-Co (Cofcc) occurs as well as their transformation to α-Co (Cohcp) particles active in the syngas conversion to C5+ hydrocarbons. This can explain the highest specific yield of C5+ hydrocarbons on a cermet with the lowest Co/Zr ratio.

Co and Co3O4 in the Hydrolysis of Boron-Containing Hydrides: H2O Activation on the Metal and Oxide Active Centers.

This work focuses on the comparison of H2 evolution in the hydrolysis of boron-containing hydrides (NaBH4, NH3BH3, and (CH2NH2BH3)2) over the Co metal catalyst and the Co3O4-based catalysts. The Co3O4 catalysts were activated in the reaction medium, and a small amount of CuO was added to activate Co3O4 under the action of weaker reducers (NH3BH3, (CH2NH2BH3)2). The high activity of Co3O4 has been previously associated with its reduced states (nanosized CoBn). The performed DFT modeling shows that activating water on the metal-like surface requires overcoming a higher energy barrier compared to hydride activation. The novelty of this study lies in its focus on understanding the impact of the remaining cobalt oxide phase. The XRD, TPR H2, TEM, Raman, and ATR FTIR confirm the formation of oxygen vacancies in the Co3O4 structure in the reaction medium, which increases the amount of adsorbed water. The kinetic isotopic effect measurements in D2O, as well as DFT modeling, reveal differences in water activation between Co and Co3O4-based catalysts. It can be assumed that the oxide phase serves not only as a precursor and support for the reduced nanosized cobalt active component but also as a key catalyst component that improves water activation.

The PB1 and the ZZ domain of the autophagy receptor p62/SQSTM1 regulate the interaction of p62/SQSTM1 with the autophagosome protein LC3B.

Autophagy is a highly conserved cellular process that allows degradation of large macromolecules. p62/SQSTM1 is a key adaptor protein that interacts both with material to be degraded and with LC3 at the autophagosome, enabling degradation of cargos such as protein aggregates, lipid droplets and damaged organelles by selective autophagy. Dysregulation of autophagy contributes to the pathogenesis of many diseases. In this study, we investigated if the interaction of p62/SQSTM1 with LC3B could be regulated. We purified full-length p62/SQSTM1 and established an in vitro assay that measures the interaction with LC3B. We used the assay to determine the role of the different domains of p62/SQSTM1 in the interaction with LC3B. We identified a mechanism of regulation of p62/SQSTM1 where the ZZ and the PB1 domains regulate the exposure of the LIR-sequence to enable or inhibit the interaction with LC3B. A mutation to mimic the phosphorylation of a site on the ZZ domain leads to increased interaction with LC3B. Also, a small compound that binds to the ZZ domain enhances interaction with LC3B. Dysregulation of these mechanisms in p62/SQSTM1 could have implications for diseases where autophagy is affected. In conclusion, our study highlights the regulated nature of p62/SQSTM1 and its ability to modulate the interaction with LC3B through a LIR-sequence Accessibility Mechanism (LAM). Furthermore, our findings suggest the potential for pharmacological modulation of the exposure of LIR, paving the way for future therapeutic strategies.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

Approaches to the design of efficient and stable catalysts for biofuel reforming into syngas: doping the mesoporous MgAl2O4 support with transition metal cations.

The mesoporous MgAl2O4 support is promising for the design of efficient and stable to coking catalysts for natural gas and biofuel reforming into syngas. This work aims at doping this support with transition metal cations (Fe, Cr, Ti) to prevent the incorporation of Ni and rare-earth cations (Pr, Ce, Zr), loaded by impregnation, into its lattice along with providing additional sites for CO2 activation required to prevent coking. Doped MgAl1.9Me0.1O4 (Me = Fe, Ti, Cr) mesoporous supports prepared by the one-pot evaporation-induced self-assembly method with Pluronic P123 triblock copolymers were single-phase spinels. Their specific surface area varies in the range of 115-200 m2 g-1, decreasing to 90-110 m2 g-1 after successive addition of the supporting nanocomposite active component 10 wt% Pr0.3Ce0.35Zr0.35O2 + (5 wt% Ni + 1% Ru) by impregnation. Mössbauer spectroscopy for iron-doped spinels confirmed the spatially uniform distribution of Fe3+ cations in the lattice without clustering being mainly located at the octahedral positions. Fourier-transform infrared spectroscopy of the adsorbed CO molecules was performed to estimate the surface density of metal sites. In methane dry reforming, the positive effect of MgAl2O4 support doping was observed from both a higher turn-over frequency as compared with the catalyst on the undoped support as well as the highest efficient first-order rate constant for the Cr-doped catalyst as compared with published data for a variety of Ni-containing catalysts based on the alumina support. In the reaction of ethanol steam reforming, the efficiency of catalysts on the doped supports is comparable, while exceeding that of Ni-containing supported catalysts reported in the literature. Coking stability was provided by a high oxygen mobility in the surface layers estimated by the oxygen isotope heteroexchange with C18O2. A high efficiency and coking stability were demonstrated in the reactions of methane dry reforming and ethanol dry and steam reforming in concentrated feeds for the honeycomb catalyst with a nanocomposite active component on the Fe-doped MgAl2O4 support loaded on the FeCrAl-alloy foil substrate.

Catalytic Activity of LaFe0.4Ni0.6O3/CeO2 Composites in CO and CH4 Oxidation Depending on Their Preparation Conditions.

LaFe0.4Ni0.6O3/CeO2 (1:1) two-phase composite materials were prepared by mechanochemical (MC) and Pechini routes. The catalytic properties of the composites in methane and CO oxidation reactions strongly depend on their preparation conditions. In low-temperature (<600 °C) catalytic CO oxidation the composites demonstrate a higher activity compared with LaFe0.4Ni0.6O3 perovskite. The highest activity was observed for the composite prepared by mechanical treatment of perovskite and fluorite precursors. There is a correlation between activity and the content of weakly bound surface oxygen species. Catalytic activity in high-temperature (>750 °C) catalytic methane oxidation correlates with the reducibility of samples. The highest activity was observed for the composite prepared by the one-pot Pechini route with higher reducibility of the sample up to 600 °C.

Toward effective Atg8-based ATTECs: Approaches and perspectives.

Induction of Atg8-family protein (LC3/GABARAP proteins in human) interactions with target proteins of interest by proximity-inducing small molecules offers the possibility for novel targeted protein degradation approaches. However, despite intensive screening campaigns during the last 5 years, no potent ligands for LC3/GABARAPs have been developed, rendering this approach largely unexplored and unsuitable for therapeutic exploitation. In this Viewpoint, we analyze the reported attempts identifying LC3/GABARAP inhibitors and provide our own point of view why no potent inhibitors have been found. Additionally, we designate reasonable directions for the identification of potent and probably selective LC3/GABARAP inhibitors for alternative therapeutic applications.

Atg8 family proteins, LIR/AIM motifs and other interaction modes.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

Effect of Ce/Zr Composition on Structure and Properties of Ce1-xZrxO2 Oxides and Related Ni/Ce1-xZrxO2 Catalysts for CO2 Methanation.

Ce1-xZrxO2 oxides (x = 0.1, 0.25, 0.5) prepared via the Pechini route were investigated using XRD analysis, N2 physisorption, TEM, and TPR in combination with density functional theory calculations. The Ni/Ce1-xZrxO2 catalysts were characterized via XRD analysis, SEM-EDX, TEM-EDX, and CO chemisorption and tested in carbon dioxide methanation. The obtained Ce1-xZrxO2 materials were single-phase solid solutions. The increase in Zr content intensified crystal structure strains and favored the reducibility of the Ce1-xZrxO2 oxides but strongly affected their microstructure. The catalytic activity of the Ni/Ce1-xZrxO2 catalysts was found to depend on the composition of the Ce1-xZrxO2 supports. The detected negative effect of Zr content on the catalytic activity was attributed to the decrease in the dispersion of the Ni0 nanoparticles and the length of metal-support contacts due to the worsening microstructure of Ce1-xZrxO2 oxides. The improvement of the redox properties of the Ce1-xZrxO2 oxide supports through cation modification can be negated by changes in their microstructure and textural characteristics.

Binding Adaptation of GS-441524 Diversifies Macro Domains and Downregulates SARS-CoV-2 de-MARylation Capacity.

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.

A Toolbox for the Generation of Chemical Probes for Baculovirus IAP Repeat Containing Proteins.

E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.

CO2 Methanation: Nickel-Alumina Catalyst Prepared by Solid-State Combustion.

The development of solvent-free methods for the synthesis of catalysts is one of the main tasks of green chemistry. A nickel-alumina catalyst for CO2 methanation was synthesized by solid-state combustion method using hexakis-(imidazole) nickel (II) nitrate complex. Using X-ray Powder Diffraction (XRD), Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and Hydrogen temperature-programmed reduction (H2-TPR), it was shown that the synthesized catalyst is characterized by the localization of easily reduced nickel oxide on alumina surface. This provided low-temperature activation of the catalyst in the reaction mixture containing 4 vol% CO2. In addition, the synthesized catalyst had higher activity in low-temperature CO2 methanation compared to industrial NIAP-07-01 catalyst, which contained almost three times more hard-to-reduce nickel-aluminum spinel. Thus, the proposed approaches to the synthesis and activation of the catalyst make it possible to simplify the catalyst preparation procedure and to abandon the use of solvents, which must be disposed of later on.

A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1.

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

Demonstrating Ligandability of the LC3A and LC3B Adapter Interface.

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure-activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).

Characterization of a natural variant of human NDP52 and its functional consequences on mitophagy.

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.

Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond.

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.

Understanding the geobiology of the terminal Ediacaran Khatyspyt Lagerstätte (Arctic Siberia, Russia).

The Khatyspyt Lagerstätte (~544 Ma, Russia) provides a valuable window into late Ediacaran Avalon-type ecosystems with rangeomorphs, arboreomorphs, and mega-algae. Here, we tackle the geobiology of this Lagerstätte by the combined analysis of paleontological features, sedimentary facies, and lipid biomarkers. The Khatyspyt Formation was deposited in carbonate ramp environments. Organic matter (0.12-2.22 wt.% TOC) displays characteristic Ediacaran biomarker features (e.g., eukaryotic steranes dominated by the C29 stigmastane). Some samples contain a putative 2-methylgammacerane that was likely sourced by ciliates and/or bacteria. 24-isopropylcholestane and 26-methylstigmastane are consistently scarce (≤0.4% and ≤0.2% of ∑C27-30 regular steranes, respectively). Thus, Avalon-type organisms occupied different niches than organisms capable of directly synthesizing C30 sterane precursors among their major lipids. Relative abundances of eukaryotic steranes and bacterial hopanes (sterane/hopane ratios = 0.07-0.30) demonstrate oligotrophic and bacterially dominated marine environments, similar to findings from other successions with Ediacara-type fossils. Ediacara-type fossils occur in facies characterized by microbial mats and biomarkers indicative for a stratified marine environment with normal-moderate salinities (moderate-high gammacerane index of 2.3-5.7; low C35 homohopane index of 0.1-0.2). Mega-algae, in contrast, are abundant in facies that almost entirely consist of allochthonous event layers. Biomarkers in these samples indicate a non-stratified marine environment and normal salinities (low gammacerane index of 0.6-0.8; low C35 homohopane index of 0.1). Vertical burrowers occur in similar facies but with biomarker evidence for stratification in the water column or around the seafloor (high gammacerane index of 5.6). Thus, the distribution of macro-organisms and burrowers was controlled by various, dynamically changing environmental factors. It appears likely that dynamic settings like the Khatyspyt Lagerstätte provided metabolic challenges for sustenance and growth which primed eukaryotic organisms to cope with changing environmental habitats, allowing for a later diversification and expansion of complex macroscopic life in the marine realm.

Ubiquitination in the ERAD Process.

In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.