Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions.

Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a ß-grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.

Total chemical synthesis of murine ISG15 and an activity-based probe with physiological binding properties.

The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. Pilot experiments showed that the synthetic proteins were properly folded and recognized by endogenous enzymes. Our synthesis strategy allows the generation of other mISG15-based probes and reagents that can accelerate the research in this field.

Total Chemical Synthesis of All SUMO-2/3 Dimer Combinations.

One hallmark of protein chemical synthesis is its capacity to access proteins that living systems can hardly produce. This is typically the case for proteins harboring post-translational modifications such as ubiquitin or ubiquitin-like modifiers. Various methods have been developed for accessing polyubiquitin conjugates by semi- or total synthesis. Comparatively, the preparation of small-ubiquitin-like modifier (SUMO) conjugates, and more particularly of polySUMO scaffolds, is much less developed. We describe hereinafter a synthetic strategy for accessing all SUMO-2/3 dimer combinations.

De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains.

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.

Exploring the Functional Consequences of Protein Backbone Alteration in Ubiquitin through Native Chemical Ligation.

Ubiquitin (Ub) plays critical roles in myriad protein degradation and signaling networks in the cell. We report herein Ub mimetics based on backbones that blend natural and artificial amino acid units. The variants were prepared by a modular route based on native chemical ligation. Biological assays show that some are enzymatically polymerized onto protein substrates, and that the resulting Ub tags are recognized for downstream pathways. These results advance the size and complexity of folded proteins mimicked by artificial backbones and expand the functional scope of such agents.

Rapid Semisynthesis of Acetylated and Sumoylated Histone Analogs.

The density and diversity of posttranslational modifications (PTMs) observed in histone proteins typically limit their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys14 or Lys56, using thiol-ene coupling chemistry. Both strategies afford multimilligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms.

Efficient Palladium-Assisted One-Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis.

The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd(II) complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one-pot fashion by native chemical ligation where the Acm moiety was used to protect the N-terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin-like protein UBL-5, which contains two native Cys residues, was accomplished through the one-pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.

Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets.

Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents.

Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.

Synthesis of N-Hydroxy Isopeptide Containing Proteins.

Chemical synthesis of a peptide-ubiquitin conjugate linked by an N-hydroxy isopeptide bond to determine what effect the N-hydroxy group has on the enzymatic hydrolysis of the isopeptide linkage by deubiquitinases is reported. This conjugate was subjected to proteolysis by UCH-L3 in the presence and absence of various metal ions, and no substantive difference in hydrolysis was seen compared to a control lacking the N-hydroxy group. The accessibility of N-hydroxy ubiquitinated substrates may find uses to study other deubiquitinases in particular those which use a zinc ion as a part of their catalytic mechanism.

Native chemical ubiquitination using a genetically incorporated azidonorleucine.

A robust chemical ubiquitination method was developed. The method employed a genetically incorporated azidonorleucine as an orthogonal lysine precursor for the installation of a Gly residue bearing an Nα-auxiliary which mediated the ligation between ubiquitin(1-75)-thioester and the target protein. To demonstrate our methodology, a model protein, K48-linked diubiquitin, was synthesized with an overall yield of 35%.

Synthesis of atypical diubiquitin chains.

Post-translational modification of proteins with ubiquitin (Ub) and Ub chains controls numerous biochemical events. Although it has been proven that all Ub-Ub linkages are formed in cells, studies have been limited for a long time to K48 and K63 chains as these can be generated biochemically. Access to the remaining (atypical) Ub-Ub chain types has been hampered by a lack of specific E2 enzymes. In this chapter we present a solution to this problem by using a native chemical ligation approach to obtain all other (i.e. K6, K11, K27, K29 and K33) diubiquitin chains.

Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine.

The dual native chemical ligation at lysine strategy was revised by replacing the acid-labile Cbz protecting group with photolabile NVOC at the 4-mercaptolysine side chain. The optimized strategy was subsequently applied to the synthesis of K48-linked diubiquitin.

Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase.

Ubiquitination is a reversible post-translational modification that regulates a myriad of eukaryotic functions. Our ability to study the effects of ubiquitination is often limited by the inaccessibility of homogeneously ubiquitinated proteins. In particular, elucidating the roles of the so-called 'atypical' ubiquitin chains (chains other than Lys48- or Lys63-linked ubiquitin), which account for a large fraction of ubiquitin polymers, is challenging because the enzymes for their biosynthesis are unknown. Here we combine genetic code expansion, intein chemistry and chemoselective ligations to synthesize 'atypical' ubiquitin chains. We solve the crystal structure of Lys6-linked diubiquitin, which is distinct from that of structurally characterized ubiquitin chains, providing a molecular basis for the different biological functions this linkage may regulate. Moreover, we profile a panel containing 10% of the known human deubiquitinases on Lys6- and Lys29-linked ubiquitin and discover that TRABID cleaves the Lys29 linkage 40-fold more efficiently than the Lys63 linkage.

N-methylcysteine-mediated total chemical synthesis of ubiquitin thioester.

Ubiquitin thioester is a key intermediate in the ubiquitylation of proteins and is formed enzymatically through the activation of alpha-COOH of ubiquitin in an ATP dependent manner using the E1 enzyme. The current methods used for the preparation of ubiquitin thioester rely on either the enzymatic machinery or on expressed protein ligation technology. In this article, we report a new chemical strategy, combining native chemical ligation and N-methylcysteine containing peptides, to chemically prepare ubiquitin thioester for the first time. The N-methylcysteine is utilized as an N-->S acyl transfer device, and in its protected form serves as a latent thioester functionality. This enabled us to trigger the formation of ubiquitin thioester subsequent to the assembly of the ubiquitin polypeptide via native chemical ligation. The synthetic ubiquitin thioester showed a similar behavior in peptide ubiquitylation to the one obtained via expression. This approach should allow for higher flexibility in the chemical manipulation of ubiquitin thioester in a wide variety of ubiquitylated peptides and proteins for structural and biochemical analysis and for the synthesis of ubiquitin chains.

Los Premios Nobel 2004 en Fisiología o Medicina y en Química: La importancia de los olores y del «Beso de la muerte» / The Nobel Prizes 2004 in Physiology or Medicine and Chemistry. The importance of the scents and the «Kiss of the death»

Como introducción a la sesión científica que esta Real Academia dedica a los Premios Nobel 2004 en Fisiología o Medicina, se hace un breve comentario al trabajo de los doctores Axel y Buck sobre los receptores olfativos y la organización del sistema olfativo dentro de un marco de referencia de la Genética del Comportamiento Humano de la percepción de los sentidos. El trabajo seminal de Axel yBuck de 1991 permitió caracterizar 18 miembros diferentes de una familia multigénica que codifica para siete dominios proteicos transmembrana cuya expresión está restringida al epitelio olfativo. Posteriormente, ambos dedicaron sus esfuerzos en clarificar el sistema del olfato desde el nivel molecular al de organización topográficacelular. En cuanto al Premio Nobel 2004 en Química, se glosa brevemente el trabajo delos doctores Ciechanover, Hershko y Rose, sobre la degradación de las proteínas mediatizada por ubiquitina. La degradación de las proteínas por los proteosomas responde a un proceso altamente regulado que comienza con su marcado molecular:el «beso de la muerte» de las ubiquitinas

As an introduction to the scientific session that the National Royal Academy of Pharmacy devotes to the Nobel Prize in Physiology or Medicine 2004, a brief commentary is made about the investigations carried out by Drs. Axel and Buck on odorant receptors and the organization of the olfactory system. In their pivotal work of 1991, they described a very large multigene family for odorant receptors, characterizing 18 members that encode seven transmembrane domain proteins whose expression is restricted to the olfactory epithellium. Afterwards, working in dependently, they clarify the olfactory system, from the molecular level to the organization of the cells involved. In regard to the Nobel Prize in Chemistry 2004, the investigations of Drs. Ciechanover, Hersko and Rose on the ubiquitin-mediated protein degradation is briefly commented. The protein degradation by the proteasomes is not indiscriminated but hightly regulated. The controlled process begins when the protein is labeled by y ubiquitin: it is the «kiss of death»

A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14.

A C-terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site-directed probe that irreversibly modifies a subset of Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [(125)I]UbVS labeling with N-ethylmaleimide and Ub aldehyde, but also by genetic analysis. [(125)I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [(125)I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S-associated USP14 with [(125)I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.