Modification of N-terminal α-amine of proteins via biomimetic ortho-quinone-mediated oxidation.

Naturally abundant quinones are important molecules, which play essential roles in various biological processes due to their reduction potential. In contrast to their universality, the investigation of reactions between quinones and proteins remains sparse. Herein, we report the development of a convenient strategy to protein modification via a biomimetic quinone-mediated oxidation at the N-terminus. By exploiting unique reactivity of an ortho-quinone reagent, the α-amine of protein N-terminus is oxidized to generate aldo or keto handle for orthogonal conjugation. The applications have been demonstrated using a range of proteins, including myoglobin, ubiquitin and small ubiquitin-related modifier 2 (SUMO2). The effect of this method is further highlighted via the preparation of a series of 17 macrophage inflammatory protein 1ß (MIP-1ß) analogs, followed by preliminary anti-HIV activity and cell viability assays, respectively. This method offers an efficient and complementary approach to existing strategies for N-terminal modification of proteins.

Nanostructured recombinant cytokines: A highly stable alternative to short-lived prophylactics.

Cytokines have been widely used as adjuvants and therapeutic agents in treatments of human diseases. Despite their recognized potential as drugs, the medical use of cytokines has considerable drawbacks, mainly related to their low stability and short half-life. Such intrinsic limitations imply the administration of high doses, often prompting toxicity, undesirable side effects and greater production costs. Here, we describe a new category of mechanically stable nanostructured cytokines (TNFα and CCL4/MIP-1ß) that resist harsh physicochemical conditions in vitro (pH and temperature), while maintaining functionality. These bio-functional materials are produced in recombinant cell factories through cost-effective and fully scalable processes. Notably, we demonstrate their prophylactic potential in vivo showing they protect zebrafish from a lethal infection by Pseudomonas aeruginosa.

Modulation of Hematopoietic Chemokine Effects In Vitro and In Vivo by DPP-4/CD26.

Dipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1ß, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.

Structures of human CCL18, CCL3, and CCL4 reveal molecular determinants for quaternary structures and sensitivity to insulin-degrading enzyme.

CC chemokine ligands (CCLs) are 8- to 14-kDa signaling proteins involved in diverse immune functions. While CCLs share similar tertiary structures, oligomerization produces highly diverse quaternary structures that protect chemokines from proteolytic degradation and modulate their functions. CCL18 is closely related to CCL3 and CCL4 with respect to both protein sequence and genomic location, yet CCL18 has distinct biochemical and biophysical properties. Here, we report a crystal structure of human CCL18 and its oligomerization states in solution based on crystallographic and small-angle X-ray scattering analyses. Our data show that CCL18 adopts an α-helical conformation at its N-terminus that weakens its dimerization, explaining CCL18's preference for the monomeric state. Multiple contacts between monomers allow CCL18 to reversibly form a unique open-ended oligomer different from those of CCL3, CCL4, and CCL5. Furthermore, these differences hinge on proline 8, which is conserved in CCL3 and CCL4 but is replaced by lysine in human CCL18. Our structural analyses suggest that a mutation of proline 8 to alanine stabilizes a type 1 ß-turn at the N-terminus of CCL4 to prevent dimerization but prevents dimers from making key contacts with each other in CCL3. Thus, the P8A mutation induces depolymerization of CCL3 and CCL4 by distinct mechanisms. Finally, we used structural, biochemical, and functional analyses to unravel why insulin-degrading enzyme degrades CCL3 and CCL4 but not CCL18. Our results elucidate the molecular basis for the oligomerization of three closely related CC chemokines and suggest how oligomerization shapes CCL chemokine function.

Structural insights into the interaction between a potent anti-inflammatory protein, viral CC chemokine inhibitor (vCCI), and the human CC chemokine, Eotaxin-1.

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1ß (macrophage inflammatory protein-1ß) complex indicated that vCCI uses negatively charged residues in ß-sheet II to interact with positively charged residues in the MIP-1ß N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1ß complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1ß, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.

Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme.

Macrophage inflammatory protein-1 (MIP-1), MIP-1α (CCL3) and MIP-1ß (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1α and MIP-1ß form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1α and MIP-1ß form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1α, thus depolymerization mutations enhance MIP-1α to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1α ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.

Deletion of the p107/p130-binding domain of Mip130/LIN-9 bypasses the requirement for CDK4 activity for the dissociation of Mip130/LIN-9 from p107/p130-E2F4 complex.

Mip130/LIN-9 is part of a large complex that includes homologs of the Drosophila dREAM (drosophila RB-like, E2F, and Myb) and C. elegans DRM complexes. This complex also includes proteins such as Mip40/LIN-37, Mip120/LIN-54, and LIN-52. In mammalian cells, Mip130/LIN-9 specifically associates with the p107/p130-E2F4 repressor complex in G0/G1 and with B-Myb in S-phase. However, little is known about how the transition occurs and whether Mip130/LIN-9 contributes to the repressor effect of p107/p130. In this report, we demonstrate that Mip130/LIN-9, Mip40/LIN-37, Mip120/LIN-54, and Sin3b form a core complex, the Mip Core Complex or LIN Complex (MCC/LINC), which is detectable in all phases of the cell cycle. This complex specifically recruits transcriptional repressors such as p107, p130, E2F4 and HDAC1 in G0/G1, and B-Myb in S-phase. Importantly, we provide strong evidence that the transition between repressors and activators of transcription is mediated by CDK4, through the phosphorylation of the pocket proteins, p107 and p130. The requirement for CDK4 activity is bypassed by the deletion of the first 84 amino acids (Mip130/LIN-9(Delta84)), since this mutant is unable to interact with p107/p130 in G0/G1, while maintaining its association with B-Myb. Importantly, the Mip130/LIN-9(Delta84) allele rescues the low expression of G1/S genes observed in CDK4(-/-) MEFs demonstrating that Mip130/LIN-9 contributes to the repression of these E2F-regulated genes in G0/G1.

Conservation of unfavorable sequence motifs that contribute to the chemokine quaternary state.

In the chemokine family, we characterize two examples of evolutionarily conserved unfavorable sequence motifs that affect quaternary structure. In contrast to the straightforward action of favorable sequences, these unfavorable motifs produce interactions disfavoring one outcome to indirectly promote another one but should not be confused with the broad sampling produced by negative selection and/or design. To identify such motifs, we developed a statistically validated computational method combining structure and phylogeny. This approach was applied in an analysis of the alternate forms of homodimerization exhibited in the chemokine family. While the chemokine family exhibits the same tertiary fold, members of certain subfamilies, including CXCL8, form a homodimer across the beta1 strand whereas members of other subfamilies, including CCL4 and CCL2, form a homodimer on the opposite side of the chemokine fold. These alternate dimerization states suggest that CCL4 and CCL2 contain specific sequences that disfavor CXCL8 dimerization. Using our computational approach, we identified two evolutionarily conserved sequence motifs in the CC subfamilies: a drastic two-residue deletion (DeltaRV) and a simple point mutation (V27R). Cloned into the CXCL8 background, these two motifs were experimentally proven to confer a monomeric state. NMR analyses indicate that these variants are structured in solution and retain the chemokine fold. Structurally, the motifs retain a chemokine tertiary fold while introducing unfavorable quaternary interactions that inhibit CXCL8 dimerization. In demonstrating the success of our computational method, our results argue that these unfavorable motifs have been evolutionarily conserved to specifically disfavor one dimerization state and, as a result, indirectly contribute to favoring another.