Highly specialized ubiquitin-like modifications: shedding light into the UFM1 enigma

Post-translational modification with Ubiquitin-like proteins represents a complex signaling language regulating virtually every cellular process. Among these post-translational modifiers is Ubiquitin-fold modifier (UFM1), which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Originally identified to be involved embryonic development, its biological function remains enigmatic. Recent research reveals that UFM1 regulates a variety of cellular events ranging from DNA repair to autophagy and ER stress response implicating its involvement in a variety of diseases. Given the contribution of UFM1 to numerous pathologies, the enzymes of the UFM1 cascade represent attractive targets for pharmacological inhibition. Here we discuss the current understanding of this cryptic post-translational modification especially its contribution to disease as well as expand on the unmet needs of developing chemical and biochemical tools to dissect its role.Chemical Immunolog

Targeting TRIM proteins: a quest towards drugging an emerging protein class

The ubiquitylation machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signaling pathways. As a consequence, its dysregulation is linked to diseases including cancer, neurodegeneration, and autoimmunity. With this review, we aim to highlight the therapeutic potential of targeting E3 ligases, with a special focus on an emerging class of RING ligases, named tri-partite motif (TRIM) proteins, whose role as targets for drug development is currently gaining pharmaceutical attention. TRIM proteins exert their catalytic activity as scaffolds involved in many protein-protein interactions, whose multidomains and adapter-like nature make their druggability very challenging. Herein, we give an overview of the current understanding of this class of single polypeptide RING E3 ligases and discuss potential targeting options.Chemical Immunolog

The effect of a high-fat diet on brain plasticity, inflammation and cognition in female ApoE4-knockin and ApoE-knockout mice

Apolipoprotein E4 (ApoE4), one of three common isoforms of ApoE, is a major risk factor for late-onset Alzheimer disease (AD). ApoE-deficient mice, as well as mice expressing human ApoE4, display impaired learning and memory functions and signs of neurodegeneration. Moreover, ApoE protects against high-fat (HF) diet induced neurodegeneration by its role in the maintenance of the integrity of the blood-brain barrier. The influence of a HF diet on the progression of AD-like cognitive and neuropathological changes was assessed in wild-type (WT), human ApoE4 and ApoE-knockout (ApoE-/-) mice to evaluate the modulatory role of ApoE in this process. From 12 months of age, female WT, ApoE4, and ApoE-/- mice were fed either a standard or a HF diet (19% butter, 0.5% cholate, 1.25% cholesterol) throughout life. At 15 months of age mice performed the Morris water maze, evaluating spatial learning and memory. ApoE-/- showed increased spatial learning compared to WT mice (p = 0.009). HF diet improved spatial learning in WT mice (p = 0.045), but did not affect ApoE4 and ApoE-/- mice. Immunohistochemical analyses of the hippocampus demonstrated increased neuroinflammation (CD68) in the cornu ammonis 1 (CA1) region in ApoE4 (p = 0.001) and in ApoE-/- (p = 0.032) mice on standard diet. HF diet tended to increase CD68 in the CA1 in WT mice (p = 0.052), while it decreased in ApoE4 (p = 0.009), but ApoE-/- remained unaffected. A trend towards increased neurogenesis (DCX) was found in both ApoE4 (p = 0.052) and ApoE-/- mice (p = 0.068). In conclusion, these data suggest that HF intake induces different effects in WT mice compared to ApoE4 and ApoE-/- with respect to markers for cognition and neurodegeneration. We propose that HF intake inhibits the compensatory mechanisms of neuroinflammation and neurogenesis in aged female ApoE4 and ApoE-/- mice

Exploring the versatility of the covalent thiol-alkyne reaction with substituted propargyl warheads: a deciding role for the cysteine protease

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CD(WT) and catalytically inactive mutant USP16CD(C205A). Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.Proteomic

Generation of the UFM1 Toolkit for Profiling UFM1-Specific Proteases and Ligases

Chemical Immunolog

Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates(1,2). However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies(3-7). Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.Chemical Immunolog

Total Chemical Synthesis of SUMO and SUMO-Based Probes for Profiling the Activity of SUMO-Specific Proteases

Chemical Immunolog

Strategy for development of site-specific ubiquitin antibodies

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.Chemical Immunolog

Design, Synthesis and Evaluation of Macrocyclic Antifungal Peptides

Fungi are increasingly recognised as major additional pathogens in already critically ill patients. Invasive fungal infections represent a growing threat and over the past two decades the incidence and diversity of fungal infections has increased enormously, especially among immunocompromised patients and patients hospitalized with serious underlying diseases. Resistance against and toxicity of the current antifungal agents underscores the urgent need for development of new antifungal compounds preferentially acting on novel targets. The echinocandins represent the most recent contribution to the arsenal of antifungal compounds that have reached the market in the last decade and act on a novel target. The echinocandins interfere with the synthesis of β-(1,3)-glucan, the major component of the framework of the fungal cell wall, by inhibiting the β-(1,3)-glucan synthase enzyme. They exhibit the most promising pharmacological, and toxicological profiles of the current arsenal of antifungal agents, as 1,3-β-glucan is only found in fungi and not in mammalian cells. As a consequence fewer toxic side effects are observed, as compared to the other classes of antifungal agents. Moreover, the clinically approved agents for this class have rarely shown fungal resistance selection. Despite these advantages, the echinocandin class of compounds has its own limitations. Firstly, due to their semisynthetic nature, they are costly. Secondly their mechanism of action is still unknown. The research described in this Thesis aims at the exploration of the crucial structural features necessary for the antifungal activity of the echinocandin compounds, by synthesizing new derivatives for structure activity studies. This information can be used for the development of improved drugs and may contribute to elucidation of the mode of action

Design, Synthesis and Evaluation of Macrocyclic Antifungal Peptides

Fungi are increasingly recognised as major additional pathogens in already critically ill patients. Invasive fungal infections represent a growing threat and over the past two decades the incidence and diversity of fungal infections has increased enormously, especially among immunocompromised patients and patients hospitalized with serious underlying diseases. Resistance against and toxicity of the current antifungal agents underscores the urgent need for development of new antifungal compounds preferentially acting on novel targets. The echinocandins represent the most recent contribution to the arsenal of antifungal compounds that have reached the market in the last decade and act on a novel target. The echinocandins interfere with the synthesis of β-(1,3)-glucan, the major component of the framework of the fungal cell wall, by inhibiting the β-(1,3)-glucan synthase enzyme. They exhibit the most promising pharmacological, and toxicological profiles of the current arsenal of antifungal agents, as 1,3-β-glucan is only found in fungi and not in mammalian cells. As a consequence fewer toxic side effects are observed, as compared to the other classes of antifungal agents. Moreover, the clinically approved agents for this class have rarely shown fungal resistance selection. Despite these advantages, the echinocandin class of compounds has its own limitations. Firstly, due to their semisynthetic nature, they are costly. Secondly their mechanism of action is still unknown. The research described in this Thesis aims at the exploration of the crucial structural features necessary for the antifungal activity of the echinocandin compounds, by synthesizing new derivatives for structure activity studies. This information can be used for the development of improved drugs and may contribute to elucidation of the mode of action