RESUMEN
The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.
Asunto(s)
Proteínas Bacterianas , Homeostasis , Legionella pneumophila , Ubiquitina , Ubiquitinación , Ubiquitina/metabolismo , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidad , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ADP-Ribosilación , Interacciones Huésped-Patógeno , Adenosina Difosfato Ribosa/metabolismo , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Células HEK293 , Actinas/metabolismo , Células HeLaRESUMEN
The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.
RESUMEN
Deciphering ubiquitin proteoform signaling and its role in disease has been a long-standing challenge in the field. The effects of ubiquitin modifications, its relation to ubiquitin-related machineries, and its signaling output has been particularly limited by its reconstitution and means of characterization. Advances in genetic code expansion have contributed towards addressing these challenges by precision incorporation of unnatural amino acids through site selective codon suppression. This review discusses recent advances in studying the 'writers', 'readers', and 'erasers' of the ubiquitin code using genetic code expansion. Highlighting strategies towards genetically encoded protein ubiquitination, ubiquitin phosphorylation, acylation, and finally surveying ubiquitin interactions, we strive to bring attention to this unique approach towards addressing a widespread proteoform problem.
Asunto(s)
Código Genético , Ubiquitina , Ubiquitinación , Ubiquitina/metabolismo , Ubiquitina/genética , Humanos , FosforilaciónRESUMEN
ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.
Asunto(s)
Legionella , Ubiquitina , Animales , Ubiquitina/metabolismo , Legionella/metabolismo , Drosophila melanogaster/metabolismo , ADP-Ribosilación , Adenosina Difosfato Ribosa/metabolismo , Hidrolasas/metabolismoRESUMEN
Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.
Asunto(s)
Neoplasias , Ubiquitina Tiolesterasa , Humanos , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Mama , Complejo de la Endopetidasa ProteasomalRESUMEN
This work introduces a simplified deposition procedure for multidimensional (2D/3D) perovskite thin films, integrating a phenethylammonium chloride (PEACl)-treatment into the antisolvent step when forming the 3D perovskite. This simultaneous deposition and passivation strategy reduces the number of synthesis steps while simultaneously stabilizing the halide perovskite film and improving the photovoltaic performance of resulting solar cell devices to 20.8%. Using a combination of multimodal in situ and additional ex situ characterizations, it is demonstrated that the introduction of PEACl during the perovskite film formation slows down the crystal growth process, which leads to a larger average grain size and narrower grain size distribution, thus reducing carrier recombination at grain boundaries and improving the device's performance and stability. The data suggests that during annealing of the wet film, the PEACl diffuses to the surface of the film, forming hydrophobic (quasi-)2D structures that protect the bulk of the perovskite film from humidity-induced degradation.
RESUMEN
A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.
Asunto(s)
Dominio Catalítico , Cisteína , Enfermedades Neurodegenerativas , Ubiquitina Tiolesterasa , Humanos , Sitios de Unión/genética , Cisteína/química , Cisteína/genética , Cinética , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Dispersión del Ángulo Pequeño , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/genética , Difracción de Rayos X , Enfermedades Neurodegenerativas/genéticaRESUMEN
Macrocyclic peptides represent promising scaffolds for chemical tools and potential therapeutics. Synthetic methods for peptide macrocyclization are often hampered by C-terminal epimerization and oligomerization, leading to difficult scalability. While chemical strategies to circumvent this issue exist, they often require specific amino acids to be present in the peptide sequence. Herein, we report the characterization of Ulm16, a peptide cyclase belonging to the penicillin-binding protein-type class of thioesterases that catalyze head-to-tail macrolactamization of nonribosmal peptides. Ulm16 efficiently cyclizes various nonnative peptides ranging from 4 to 6 amino acids with catalytic efficiencies of up to 3 × 106 M-1 s-1. Unlike many previously described homologs, Ulm16 tolerates a variety of C- and N-terminal amino acids. The crystal structure of Ulm16, along with modeling of its substrates and site-directed mutagenesis, allows for rationalization of this wide substrate scope. Overall, Ulm16 represents a promising tool for the biocatalytic production of macrocyclic peptides.
Asunto(s)
Aminoácidos , Péptidos , Proteínas de Unión a las Penicilinas/metabolismo , Ciclización , Péptidos/química , Biocatálisis , Aminoácidos/metabolismo , Péptidos CíclicosRESUMEN
ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.
Asunto(s)
ADP Ribosa Transferasas , Proteínas Bacterianas , Modelos Moleculares , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , ADP-Ribosilación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chromobacterium/química , Chromobacterium/enzimología , Chromobacterium/genética , Cristalografía por Rayos X , NAD/química , NAD/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Especificidad por Sustrato , Ubiquitina/metabolismoRESUMEN
Interfaces in perovskite solar cells play a crucial role in their overall performance, and therefore, detailed fundamental studies are needed for a better understanding. In the case of the classical n-i-p architecture, TiO2 is one of the most used electron-selective layers and can induce chemical reactions that influence the performance of the overall device stack. The interfacial properties at the TiO2/perovskite interface are often neglected, owing to the difficulty in accessing this interface. Here, we use X-rays of variable energies to study the interface of (compact and mesoporous) TiO2/perovskite in such a n-i-p architecture. The X-ray photoelectron spectroscopy and X-ray absorption spectroscopy methods show that the defect states present in the TiO2 layer are passivated by a chemical interaction of the perovskite precursor solution during the formation of the perovskite layer and form an organic layer at the interface. Such passivation of intrinsic defects in TiO2 removes charge recombination centers and shifts the bands upward. Therefore, interface defect passivation by oxidation of Ti3+ states, the organic cation layer, and an upward band bending at the TiO2/perovskite interface explain the origin of an improved electron extraction and hole-blocking nature of TiO2 in the n-i-p perovskite solar cells.
RESUMEN
Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB-Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB-Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.
Asunto(s)
Cisteína , Ubiquitina , Ubiquitina/química , Ubiquitinación , Dominios Proteicos , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismoRESUMEN
BACKGROUND: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). METHODS: We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. RESULTS: By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. CONCLUSIONS: Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro. Video Abstract.
Asunto(s)
Lipopolisacáridos , Proteoma , Lipopolisacáridos/farmacología , Procesamiento Proteico-Postraduccional , Fosforilación , MacrófagosRESUMEN
Background. Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). Methods. We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We integrated this method into a mass spectrometry-based multi-PTM pipeline to simultaneously analyze carbamylated and acetylated peptides in addition to phosphopeptides were enriched by sequential immobilized-metal affinity chromatography. Results. By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. Conclusions Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro .
RESUMEN
The most efficient and stable perovskite solar cells (PSCs) are made from a complex mixture of precursors. Typically, to then form a thin film, an extreme oversaturation of the perovskite precursor is initiated to trigger nucleation sites, e.g., by vacuum, an airstream, or a so-called antisolvent. Unfortunately, most oversaturation triggers do not expel the lingering (and highly coordinating) dimethyl sulfoxide (DMSO), which is used as a precursor solvent, from the thin films; this detrimentally affects long-term stability. In this work, (the green) dimethyl sulfide (DMS) is introduced as a novel nucleation trigger for perovskite films combining, uniquely, high coordination and high vapor pressure. This gives DMS a universal scope: DMS replaces other solvents by coordinating more strongly and removes itself once the film formation is finished. To demonstrate this novel coordination chemistry approach, MAPbI3 PSCs are processed, typically dissolved in hard-to-remove (and green) DMSO achieving 21.6% efficiency, among the highest reported efficiencies for this system. To confirm the universality of the strategy, DMS is tested for FAPbI3 as another composition, which shows higher efficiency of 23.5% compared to 20.9% for a device fabricated with chlorobenzene. This work provides a universal strategy to control perovskite crystallization using coordination chemistry, heralding the revival of perovskite compositions with pure DMSO.
RESUMEN
Ubiquitin (Ub) proteoforms control nearly every aspect of eukaryotic cell biology through their diversity. Inspired by the widely used Ub C-terminal electrophiles (Ub-E), here we report the identification of multivalent binding of Ub with deubiquitylating enzymes (Dubs) using genetic code expansion (GCE) and crosslinking mass spectrometry. While the Ub-Es only gather structural information with the S1 Dub sites, we demonstrate that GCE of Ub with p-benzoyl-L-phenylalanine enables identification of interaction modes beyond the S1 site with a panel of Dubs of both eukaryotic and prokaryotic origin. Collectively, this represents the next generation of Ub-based affinity probes with a unique ability to unravel Ub interaction landscapes beyond what is afforded by cysteine-based chemistries.
Asunto(s)
Células Procariotas , Ubiquitina , Ubiquitina/metabolismo , Células Procariotas/metabolismo , Células Eucariotas , UbiquitinaciónRESUMEN
The bacterial pathogen Legionella pneumophila encodes numerous effectors to manipulate host ubiquitin signaling. Recently, Warren et al. revealed the structural basis of K6-polyubiquitination recognition by Legionella deubiquitinase LotA, while validating its potential as an enzymatic tool to study linkage-specific ubiquitination. During Legionella infection, LotA counteracts valosin-containing protein (VCP) recruitment to the Legionella-containing vacuole.
Asunto(s)
Legionella pneumophila , Enfermedad de los Legionarios , Humanos , Enfermedad de los Legionarios/microbiología , Ubiquitinación , Ubiquitina/metabolismo , Legionella pneumophila/genética , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.
RESUMEN
The activity of deubiquitinases (DUBs) is tightly regulated in eukaryotes via various mechanisms. One of the regulatory strategies is substrate-induced catalytic triad rearrangement, where ubiquitin-binding helps the DUB adopt an active conformation for catalysis. The crystal structure of the apo form of such a DUB, when not bound to ubiquitin, reveals an inactive conformation of the catalytic residues, necessitating the structure of the ubiquitin-bound form to visualize the active state of the DUB. Comparing the apo and ubiquitin-bound structures reveals conformational changes leading to catalytic activation. To capture the deubiquitinase in its ubiquitin-bound form, a series of activity-based ubiquitin probes (Ub-ABPs) harboring C-terminal electrophiles were designed to react with the catalytic nucleophile of cysteine protease DUBs. The resulting covalently linked DUB-ubiquitin complex is amendable for structural studies to probe the DUB-ubiquitin interface and the potential conformational change of the DUB. Here, we present a detailed protocol for the generation and purification of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in complex with a Ub-ABP, ubiquitin-vinyl methyl ester (UbVME), and the subsequent structural analysis to characterize the catalytic state of the DUB.
Asunto(s)
Enzimas Desubicuitinizantes , Ubiquitina , Ubiquitina/metabolismo , Unión Proteica , Catálisis , Enzimas Desubicuitinizantes/metabolismoRESUMEN
Liquid metals (LMs) play a growing role in flexible electronics and connected applications. Here, LMs come into direct contact with metal electrodes thus allowing for corrosion and additional alloying, potentially compromising device stability. Nevertheless, comprehensive studies on the interfacial interaction of the materials are still sparse. Therefore, a correlated material interaction study of capillary-printed Galinstan (eutetic alloy of Ga/In/Sn) with gold surfaces and electrodes is conducted. Comprehensive application of optical microscopy, vertical scanning interferometry, scanning electron microscopy/spectroscopy, x-ray photon spectroscopy, and atomic force microscopy allow for an in depth characterization of the spreading process of LM lines on gold films, revealing the differential spread of the different LM components and the formation of intermetallic nanostructures on the surface of the surrounding gold film. A model for the growth process based on the penetration of LM along the gold film grain boundaries is proposed based on the obtained time-dependent characterization. The distribution of gold, Galinstan, and intermetallic phases in a gold wire dipped into LM is observed using X-ray nano tomography as a complementary view on the internal nanostructure. Finally, resistance measurements on LM lines connecting gold electrodes over time allow to estimate the influence on the material interaction on electronic applications.