Recombinant C1 Esterase Inhibitor Reduces Cytokine Storm in an Ex Vivo Whole Blood Model.

C1 esterase inhibitor (C1INH) is an abundant component of blood plasma (the average concentration is 250 mg/L); it is known to be involved in several biological processes, for instance, in the regulation of the coagulation system, adhesion of leukocytes on endothelial cells, and in the regulation of complement and kallikrein cascades. Lately, the role of C1INH in immunomodulation has gained considerable attention. We used an ex vivo whole blood model to examine the influence of C1INH and its mutated variants on the inflammatory cytokines interleukin (IL)-6, tumor necrosis factor-α (TNFα), and IL-1ß. The present study demonstrated for the first time that recombinant C1INH or its Seprin domain can downregulate bacterial endotoxin induced IL-6 release. We also observed that unstructured N-terminal domain of C1INH downregulates the release of IL-1ß and TNFα, but not IL-6. Our results suggest that C1INH may have therapeutic potential for treatment of inflammatory conditions.

Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO.

RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length.

Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.

Analysis of cellular SUMO and SUMO-ubiquitin hybrid conjugates.

Posttranslational modification of proteins with the small ubiquitin-related modifier (SUMO) has been implicated in many important physiological functions, including the regulation of transcription and DNA repair. In most cases, only a small fraction of the total cellular amounts of a given protein is sumoylated at a certain point in time. Sensitive detection of sumoylated forms of proteins by western blotting is, therefore, an important step in the identification and/or characterization of a protein control by sumoylation. Polysumoylated proteins are recognized and targeted to the proteasome by specific ubiquitin ligases bearing SUMO interaction motifs. Sumoylation itself is reversible by the action of desumoylating enzymes. Their activities cause a rapid loss of SUMO conjugates in most standard cell extracts. To preserve SUMO-protein conjugates, therefore, a preparation of extracts under denaturing conditions is recommended. Here, we describe the application of an alkaline lysis procedure and a western blot protocol for the analysis of SUMO conjugates in yeast and human cells. In addition, we describe the application of another extraction procedure combined with immobilized metal affinity chromatography for the analysis of ubiquitin-SUMO hybrid conjugates from yeast and human cells.