Mutant Ubiquitin Research Articles

Engineering the orthogonal ubiquitin E1-E2 pairs for identification of K27 chain linkage substrates

Ubiquitination is one kind of crucial protein post-translational modification (PTM) in eukaryotic cells, forming sorts of types of polyubiquitin chain linkages on the substrates. Ubiquitin conjugating enzymes (E2s) play an essential role in the formation of ubiquitin chains. However, the mechanism of the formation of atypical chain (such as K27) and the identification of the related substrates are not well understood. Previously we developed an orthogonal ubiquitin transfer (OUT) pathway to identify the substrates of a specific ubiquitin ligase (E3). In OUT pathway, a ubiquitin mutant (xUb) is transferred to the substrates through an engineered xE1-xE2-xE3 cascade. In this study, we reengineered a new OUT pathway for the transfer of xUb-K27, an isoform of xUb with only one lysine at Lys27 resident. The newly designed xUba1-xUbe2D2 (xE1-xE2) pairs can transfer xUb-K27 to downstream wild type E3s and form K27 linkages. Ube2D2 is known for its versatility as it forms all sorts of polyubiquitin chains on substrates. Therefore, the xE1-xE2 pairs are empowered to transfer other ubiquitin mutants, for instance, xUb-K6 and xUb-K11. The new xE1-xE2 pairs also deepen the understanding of structural information about the E1-E2 interaction, and provide further insights into the mechanism of chain formation mediated by E2.

Modeling Chromatography Binding through Molecular Dynamics Simulations with Resin Fragments.

Accurate atomistic modeling of the interactions of a chromatography resin with a solute can inform the selection of purification conditions for a product, an important problem in the biotech and pharmaceutical industries. We present a molecular dynamics simulation-based approach for the qualitative prediction of interaction sites (specificity) and retention times (affinity) of a protein for a given chromatography resin. We mimicked the resin with an unrestrained ligand composed of the resin headgroup coupled with successively larger fragments of the agarose backbone. The interactions of the ligand with the protein are simulated in an explicit solvent using the Replica Exchange Molecular Dynamics enhanced sampling approach in conjunction with Hydrogen Mass Repartitioning (REMD-HMR). We computed the ligand interaction surface from the simulation trajectories and correlated the features of the interaction surface with experimentally determined retention times. The simulation and analysis protocol were first applied to a series of ubiquitin mutants for which retention times on Capto MMC resin are available. The ubiquitin simulations helped identify the optimal ligand that was used in subsequent simulations on six proteins for which Capto MMC elution times are available. For each of the six proteins, we computed the interaction surface and characterized it in terms of a range of simulation-averaged residue-level physicochemical descriptors. Modeling of the salt concentrations required for elution with respect to the descriptors resulted in a linear fit in terms of aromaphilicity and Kyte-Doolittle hydrophobicity that was robust to outliers, showed high correlation, and correctly ranked the protein elution order. The physics-based model building approach described here does not require a large experimental data set and can be readily applied to different resins and diverse biomolecules.

AI-Powered Western Blot Interpretation: A Novel Approach to Studying the Frameshift Mutant of Ubiquitin B (UBB+1) in Schizophrenia

The application of artificial intelligence (AI) in the analysis of molecular biology data is becoming increasingly widespread. The Western Blot (WB) technique, a cornerstone in proteomic research, facilitates the identification and analysis of proteins, such as the frameshift mutant of ubiquitin B (UBB+1). In our study, we attempted to assess the potential of four different AI models—Gemini, Gemini Advanced, Microsoft Copilot, and ChatGPT 4—in the analysis of WB imagery containing UBB+1, derived from peripheral blood studies of patients suffering from schizophrenia. Participants, all male and diagnosed with schizophrenia, were recruited from the Specialist Psychiatric Care Team of Babinski Hospital in Lodz. After obtaining their informed consent, blood samples were collected and transported to the laboratory of the Department of Medical Biochemistry at the Medical University of Lodz. The samples were processed, synthesis of Ub-48UBB+1 dimers was performed, and the WB technique was applied. The result of the WB analysis, in the form of a photograph with basic labels but without a legend (JPG format), was implemented into ChatGPT 4, Microsoft Copilot, Gemini and Gemini Advanced. Following the implementation of the image, the command ‘Could you analyze the attached photo?’ was added, along with the protocol from Sample Preparation and Synthesis of Ub-48UBB+1 Dimers. The AI models effectively analyzed and interpreted WB images, with variations in their approaches and depth. Gemini excelled in detailing the WB process and biological significance of bands, while Gemini Advanced focused on specific band identification, especially Ub-48UBB+1 dimers. Microsoft Copilot provided a basic overview with less technicality, and ChatGPT 4 offered comprehensive band interpretations, linking them to patient samples and standards, thus confirming the hypothesis about the differing capabilities of these models. This discovery demonstrates the advanced capabilities of ChatGPT 4 and highlights the growing role of AI in scientific research, including the interpretation of results.

Proline Peptide Bond Isomerization in Ubiquitin Under Folding and Denaturing Conditions by Pressure-Jump NMR

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized ‘folding nucleus’. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis → trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state.

Complex Topology of Ubiquitin Chains Mediates Lysosomal Degradation of MrgC Proteins.

Activation of Mas-related G protein-coupled receptor C (MrgC) receptors relieves pain, but also leads to ubiquitination of MrgC receptors. Ubiquitination mediates MrgC receptor endocytosis and degradation. However, MrgC degradation pathways and ubiquitin-linked chain types are not known. N2a cells were treated with cycloheximide (CHX, protein synthesis inhibitor), Mg132 (proteasome inhibitor), 3-Methyladenine (3MA, autophagy lysosome inhibitor) and Chloroquine (CQ, autophagy lysosome inhibitor) to observe the half-life and degradation pathway of MrgC. The location of internalized MrgC receptors and lysosomes (Lyso-Tracker) was observed by immunofluorescence staining. N2a cells were transfected with Myc-MrgC and a series of HA-tagged ubiquitin mutants to study the ubiquitin-linked chain type of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. Autophagy lysosome inhibitors can inhibit the degradation of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. 3-MA and CQ inhibited the degradation of MrgC protein, whereas Mg-132 did not inhibit it. Partially internalized MrgC receptors were co-labeled with lysosomes. MrgC proteins have multiple topologies of ubiquitin-modified chains. As a member of the G protein-coupled receptor family, MrgC receptors can be degraded over time. The complex topology of the ubiquitin-linked chain mediates the lysosomal degradation of MrgC proteins.

Unbalanced redox status network as an early pathological even in congenital cataracts

Aims/Purpose: The lens proteome undergoes dramatic composition changes during development and maturation. Defective developmental processes lead to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early‐onset forms of lens opacity, with the remainder being of unknown aetiology. To gain a better understanding of congenital cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W‐Ub) that recapitulates many of the early pathological changes seen in human congenital cataracts.Methods: We performed MS‐based tandem‐mass‐tag quantitative proteomics (TMT), computational molecular phenotyping (CMP), high‐performance liquid chromatography (HPLC), western blotting and immunohistochemical analysis in E15, P1 and P30 control or K6W‐Ub lenses.Results: TMT identified proteins that were altered during normal lens differentiation, some of which were also altered in cataractous lenses. These included proteins in glutathione and amino acid metabolic pathways. CMP revealed that glutathione and taurine were reduced spatially altered in the K6W‐Ub cataractous lens. HPLC revealed that both the ratio GSH/GSSG and taurine, two indicators of redox status, were differentially compromised in K6W‐Ub cataractous lenses.Conclusions: Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age‐related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.Funding: Funding was provided by grants RYC 2018‐024434‐I, MINECO PID 2020‐119466RB‐I00, FUSP‐PPC‐19‐B53C4C64, NIH RO1EY026979 (to AT), USDA 8050‐51000‐089‐01S (to AT).

Unbalanced redox status network as an early pathological event in congenital cataracts

The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early-onset forms of lens opacity, with the remainder being of unknown etiology. To gain a better understanding of cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W-Ub) that recapitulates most of the early pathological changes seen in human congenital cataracts. We performed mass spectrometry-based tandem-mass-tag quantitative proteomics in E15, P1, and P30 control or K6W-Ub lenses. Our analysis identified targets that are required for early normal differentiation steps and altered in cataractous lenses, particularly metabolic pathways involving glutathione and amino acids. Computational molecular phenotyping revealed that glutathione and taurine were spatially altered in the K6W-Ub cataractous lens. High-performance liquid chromatography revealed that both taurine and the ratio of reduced glutathione to oxidized glutathione, two indicators of redox status, were differentially compromised in lens biology. In sum, our research documents that dynamic proteome changes in a mouse model of congenital cataracts impact redox biology in lens. Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age-related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.

Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination

Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3–KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease.

Water irradiation devoid pulses enhance the sensitivity of 1H,1H nuclear Overhauser effects.

The nuclear Overhauser effect (NOE) is one of NMR spectroscopy's most important and versatile parameters. NOE is routinely utilized to determine the structures of medium-to-large size biomolecules and characterize protein-protein, protein-RNA, protein-DNA, and protein-ligand interactions in aqueous solutions. Typical [1H,1H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates 1H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons. However, at high- and ultra-high magnetic fields, the excitation of the water signal during the execution of the NOESY pulse sequences may cause significant attenuation of NOE cross-peak intensities. Using an evolutionary algorithm coupled with artificial intelligence, we recently designed high-fidelity pulses [Water irrAdiation DEvoid (WADE) pulses] that elude water excitation and irradiate broader bandwidths relative to commonly used pulses. Here, we demonstrate that WADE pulses, implemented into the 2D [1H,1H] NOESY experiments, increase the intensity of the NOE cross-peaks for labile and, to a lesser extent, non-exchangeable protons. We applied the new 2D [1H,1H] WADE-NOESY pulse sequence to two well-folded, medium-size proteins, i.e., the K48C mutant of ubiquitin and the Raf kinase inhibitor protein. We observed a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments. The new WADE pulses can be easily engineered into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences to boost their sensitivity.

Localising nuclear spins by pseudocontact shifts from a single tagging site.

Ligating a protein at a specific site with a tag molecule containing a paramagnetic metal ion provides a versatile way of generating pseudocontact shifts (PCSs) in nuclear magnetic resonance (NMR) spectra. PCSs can be observed for nuclear spins far from the tagging site, and PCSs generated from multiple tagging sites have been shown to enable highly accurate structure determinations at specific sites of interest, even when using flexible tags, provided the fitted effective magnetic susceptibility anisotropy () tensors accurately back-calculate the experimental PCSs measured in the immediate vicinity of the site of interest. The present work investigates the situation where only the local structure of a protein region or bound ligand is to be determined rather than the structure of the entire molecular system. In this case, the need for gathering structural information from tags deployed at multiple sites may be queried. Our study presents a computational simulation of the structural information available from samples produced with single tags attached at up to six different sites, up to six different tags attached to a single site, and in-between scenarios. The results indicate that the number of tags is more important than the number of tagging sites. This has important practical implications, as it is much easier to identify a single site that is suitable for tagging than multiple ones. In an initial experimental demonstration with the ubiquitin mutant S57C, PCSs generated with four different tags at a single site are shown to accurately pinpoint the location of amide protons in different segments of the protein.

Rigid, Highly Reactive and Stable DOTA-like Tags Containing a Thiol-Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis*.

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons.

The synaptic expression of glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+ -impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

MCM2-7 and FANCD2 Are Required for RAD51 Function in Homologous Recombination and ICL Repair

MCM2-7 helicase is required for DNA replication and has been implicated in the maintenance of genomic instability, as more than 90% of MCM complexes are in dormant origins. Decreased levels of MCM2-7 complex on chromatin lead to genomic instability. The Fanconi anemia (FA) pathway, whose proteins are defective in the genetic disorder that entails bone marrow failure, congenital defects, and acute myeloid leukemia, is also important in maintaining genomic instability through the regulation of homologous recombinatorial repair (HRR) of interstrand cross links (ICL). FANCD2 is the central protein in the FA pathway, exhibiting monoubiquitination in response to DNA damage, and has been shown to interact with MCM2-7. Here we show that monoubiquitinated FANCD2 interacts with MCM2-7 and is required for maintenance of MCM2-7 on chromatin upon DNA interstrand crosslinking (ICL) drug mitomycin C treatment. FANCD2-MCM2-7 also interacts with RAD51, an interaction which is decreased in the absence of FANCD2 or with the expression of the ubiquitination mutant FANCD2(K561R). Also, RAD51 chromatin localization and homologous recombination (HR) function is impaired in MCM7 knockdown cells. MCM2, MCM5, or MCM7 knock down cells exhibit hypersensitive to ICL and G2/M accumulation as well, and the hypersensitivity of MCM7 knock down cells can be rescued by re-expressing MCM7. Importantly FANCD2 and MCM7 are epistatic for MMC hypersensitivity. At the levels of MCM7 knock down, we observe that cells are not defective for replication as measured by BrdU labeling and are not hypersensitive to replication inhibitor hydroxyurea (HU) but remain ICL sensitive. Using recombinant protein reconstitution in vitro, we observe MCM2-7 and FANCI-FANCD2 dependent RAD51 filament stabilization and strand exchange, which are key steps in HRR. MCM2-7 proteins defective for helicase activity and thus replication defective remain functional in these in vitro biochemical assays. On the other hand, a FANCI DNA binding mutant completely abrogates these activities, suggesting that the FANCD2-FANCI-MCM2-7-RAD51 complex requires a regulated interaction with DNA and is separable from MCM2-7 replication function. Taken together, the FA pathway, MCM2-7, and RAD51 work cooperatively in the biochemical and cell response to DNA damage ICL. DisclosuresNo relevant conflicts of interest to declare.

RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

Ubiquitination-dependent DNA damage response (DDR) signals play a critical role in the cellular choice of DNA damage repair pathways. Human DNA helicase RecQL4 participates in DNA replication and repair, and loss of RecQL4 is associated with autosomal recessive genetic disorders characterized by genomic instability features. In an earlier study, RecQL4 was isolated as a stable complex that contained two ubiquitin ligases of the N-end rule (UBR1 and UBR2). However, it is unknown whether or not RecQL4 ubiquitination status is critical for its DNA repair function. Here, we report that RecQL4 directly interacts with RNF8 (a RING finger ubiquitin E3 ligase), and both co-localize at DNA double-strand break (DSB) sites. Our findings indicate that RNF8 ubiquitinates RecQL4 protein mainly at the lysine sites of 876, 1048, and 1101, thereby facilitating the dissociation of RecQL4 from DSB sites. RecQL4 mutant at ubiquitination sites had a significantly prolonged retention at DSBs, which hinders the recruitment of its direct downstream DSB repair proteins (CtIP & Ku80). Interestingly, reduced DSB repair capacity observed in RecQL4 depleted cells was restored only by the reconstitution of wild-type RecQL4, but not the ubiquitination mutant. Additionally, RecQL4 directly interacts with WRAP53β that is known to recruit RNF8 to DSBs and WRAP53β enhances the association of RecQL4 with RNF8. WRAP53β silencing resulted in a nearly diminished recruitment of RNF8 to DSBs and in a greatly attenuated dissociation of RecQL4 from the DSB sites. Collectively, our study demonstrates that the ubiquitination event mediated by RNF8 constitutes an essential component for RecQL4’s function in DSB repair.

Probing the effects of double mutations on the versatile protein ubiquitin in Saccharomyces cerevisiae

Ubiquitin is an indispensable protein of eukaryotic origin with an extraordinarily high degree of sequence conservation. It is used to tag proteins post-translationally and the process of ubiquitination regulates the activity of the modified proteins or drives them for degradation. Double mutations produce varied effects in proteins, depending on the structural relationship of the mutated residues, their role in the overall structure and functions of a protein. Six double mutants derived from the ubiquitin mutant UbEP42, namely S20F-A46S, S20F-L50P, S20F-I61T, A46S-L50P, A46S-I61T, and L50P-I61T, have been studied here to understand how they influence the ubiquitination related functions, by analysing their growth and viability, Cdc28 levels, K-48 linked polyubiquitination, UFD pathway, lysosomal degradation, endosomal sorting, survival under heat, and antibiotic stresses. The double mutation L50P-I61T is the most detrimental, followed by S20F-I61T and A46S-I61T. The double mutations studied here, in general, make cells more sensitive than the wild type to one or the other stress. However, the excessive negative effects of L50P and I61T are compensated under certain conditions by S20F and A46S mutations. The competitive inhibition produced by these substitutions could be used to manage certain ubiquitination associated diseases.

Ubiquitination of MHC Class II by March-I Regulates Dendritic Cell Fitness.

The expression and turnover of Ag-specific peptide-MHC class II (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to efficiently activate CD4 T cells. Ubiquitination of pMHC-II by the E3 ubiquitin ligase March-I regulates surface expression and survival of pMHC-II in DCs. We now show that despite their high levels of surface pMHC-II, MHC class II (MHC-II) ubiquitination-deficient mouse DCs are functionally defective; they are poor stimulators of naive CD4 T cells and secrete IL-12 in response to LPS stimulation poorly. MHC-II ubiquitination-mutant DC defects are cell intrinsic, and single-cell RNA sequencing demonstrates that these DCs have an altered gene expression signature as compared with wild-type DCs. Curiously, these functional and gene transcription defects are reversed by activating the DCs with LPS. These results show that dysregulation of MHC-II turnover suppresses DC development and function.

The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1

Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.

Hepatitis B Core Protein Is Post-Translationally Modified through K29-Linked Ubiquitination

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

Expression of Mutant Ubiquitin and Proteostasis Impairment in Kii Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex Brains.

Kii amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder that is endemic to the Kii peninsula of Japan. The disorder is clinically characterized by a variable combination of parkinsonism, dementia, and motor neuron symptoms. Despite extensive investigations, the etiology and pathogenesis of ALS/PDC remain unclear. At the neuropathological level, Kii ALS/PDC is characterized by neuronal loss and tau-dominant polyproteinopathy. Here, we report the accumulation of several proteins involved in protein homeostasis pathways, that is, the ubiquitin-proteasome system and the autophagy-lysosome pathway, in postmortem brain tissue from a number of Kii ALS/PDC cases (n = 4). Of particular interest is the presence of a mutant ubiquitin protein (UBB+1), which is indicative of disrupted ubiquitin homeostasis. The findings suggest that abnormal protein aggregation is linked to impaired protein homeostasis pathways in Kii ALS/PDC.

Design, synthesis and evaluation of E2‐25K derived stapled peptides

AbstractStabilised peptides are now established as potential drug candidates to probe previously intractable molecular targets, such as protein‐protein interactions. Herein, we report the design and synthesis of eight short helical peptide analogues of the ubiquitin conjugating enzyme, E2‐25K, as potential antagonists of the interaction between E2‐25K and the Alzheimer's Disease (AD) associated ubiquitin mutant Ubb + 1. Biochemical evaluation revealed four putative antagonists of the Ubb + 1/E2‐25K interaction that reduced incorporation of Ubb + 1 into polyubiquitin chains in vitro, validating the potential of this approach as a therapeutic strategy.