Posttranslational modification of proteins by ubiquitin (Ub), i.e., ubiquitination, mediates a variety of cellular processes, including protein homeostasis, cell cycle, DNA repair, and viral infections. Understanding the molecular mechanism of ubiquitination in these events is the basis for unraveling its precise role in health and disease. However, the inherent complexity of Ub signaling due to the high atomic complexity of Ub conjugates, where Ub is attached to other Ub molecules and to protein substrates in various forms, imposes a major challenge for these studies. In this regard, the enzymatic approaches employed for the preparation of important Ub conjugates have severe limitations to deliver them in high homogeneity and in adequate amounts for the desired study. Recent developments in the area of chemical synthesis and semisynthesis of proteins offer great solutions to the enzymatic limitations and enabling the preparation of various Ub conjugates with precise control over the atomic structure. These conjugates significantly contribute to deciphering Ub signaling at the molecular level, and with the synthetic tools in hand, chemical biologists have become key players in efforts toward understanding the complexity of the Ub code. In this Perspective, we highlight the key contributions of these synthetic approaches and how the development of novel Ub-based reagents is greatly assisting in uncovering unknown aspects of Ub signaling. We also discuss future aspirations to address unresolved questions in this exciting area of research.

We report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO.

Chemists have been interested in the N-alkylation of a peptide bond because such a modification alters the conformation of the amide bond, interferes with hydrogen bond formation, and changes other properties of the peptide (e.g., solubility). This modification also opens the door for attaching functional groups for various applications. Nonetheless, the irreversibility of some of these modifications and the harsh conditions required for their removal currently limits the wide utility of this approach. Herein, we report applying a propargyl group for peptide bond modification at diverse junctions, which can be removed under mild and aqueous conditions via treatment with gold­(I). Considering the straightforward conditions for both the installation and removal of this group, the propargyl group provides access to the benefits of backbone N-alkylation, while preserving the ability for on-demand depropargylation and full recovery of the native amide bond. This reversible modification was found to improve solid-phase peptide synthesis as demonstrated in the chemical synthesis of NEDD8 protein, without the use of special dipeptide analogues. Also, the reported approach was found to be useful in decaging a broad range of propargyl-based protecting groups used in chemical protein synthesis. Remarkably, reversing the order of the two residues in the propargylation site resulted in rapid amide bond cleavage, which extends the applicability of this approach beyond a removable backbone modification to a cleavable linker. The easy attach/detach of this functionality was also examined in loading and releasing of biotinylated peptides from streptavidin beads.

Background: Kaposi sarcoma (KS) is a neoplasm of the endothelial cells. It often manifests with multiple vascular nodules on the skin and other organs. It is a systemic, malignant and multifactorial disease and has a variable course. There are four types: classic, endemic, iatrogenic and HIV-associated. The primary presentation on the penis and face is uncommon and is mainly observed in HIV-positive patients. Multiple treatment modalities are used including surgery, cryotherapy, electrosurgery, laser and radiation therapy.Main observation: The authors present two cases of isolated Kaposi sarcoma in HIV negative, human herpes virus 8 (HHV-8) positive non immunocompromised patients. One case with facial KS and the other one with penile KS. Both were treated surgically with no recurrence in the following 6 months of the follow up period.Conclusions: Kaposi sarcoma is rare in HIV negative patients and is associated with HHV-8 infection. Lesions are usually solitary and can be treated surgically. It should be included in the differential diagnoses of penile and facial lesions that are clinically suspecious and resistent to therapy. (J Dermatol Case Rep. 2011; 5(2): 24-26.)

An efficient native chemical ligation approach at Asp and Glu sites is reported applying a hydrazide precursor, as a peptide thioester, and allyl protection at the side chain of Asp and Glu. The allyl protection was efficiently removed, after the ligation step, using the water-soluble palladium complex [Pd(allyl)Cl]2 and glutathione within a few minutes under fully aqueous conditions.

A rapid and efficient cyclization of unprotected N-propargylated peptides using the Au(I) organometallic complex is reported. The method relies on the activation of the propargyl functionality using gold(I) to produce a new linkage with the N-terminus amine at the cyclization site. The presented method features a fast reaction rate (within 20 min), mild conditions, chemoselectivity, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains.

Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a βgrasp 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.

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