Dmitri V. Filippov

Protein-primed RNA synthesis by purified poliovirus RNA polymerase

A small protein, VPg, is covalently linked to the 5′ end of the plus-stranded poliovirus genomic RNA. Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5′ end by the methylated structure that is common to most eukaryotic mRNAs. These discoveries presented two problems. First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5′ mRNA end, has solved the first conundrum. Here we describe the resolution of the second problem. VPg is linked to the genomic RNA through the 5′-terminal uridylic acid of the RNA. We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol. Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to produce VPg-linked poly(U). Initiation of transcription of the poliovirus genome from the polyadenylated 3′ end therefore depends on VPg.

The conformation of neurotensin bound to its G protein-coupled receptor

G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13C,15N-labeled NT(8–13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a β-strand conformation. Our results provide a viable structural template for further pharmacological investigations.

Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells *

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam3CysSK4 (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG∼conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam∼conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG∼conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8+ T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity

Dendritic cells (DCs) are crucial for priming of naive CD8+ T lymphocytes to exogenous antigens, so-called “cross-priming.” We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I–peptide complexes at the cell surface.

Specific Cell-Permeable Inhibitor of Proteasome Trypsin-like Sites Selectively Sensitizes Myeloma Cells to Bortezomib and Carfilzomib

Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.

Use of benzyloxycarbonyl (Z)-based fluorophilic tagging reagents in the purification of synthetic peptides

Abstract Three novel fluorous reagents (i.e. 1 – 3 ), derived from benzyl chloroformate (Z-Cl), have been synthesized and used for the tagging of peptides prepared following a Fmoc-based solid-phase approach. It is shown that the implementation of a benzyloxycarbonyl (Z)-based fluorophilic tag facilitates purification of peptides using fluorous reverse phase chromatography.

An RNA virus hijacks an incognito function of a DNA repair enzyme

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein–RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5′-tyrosyl–DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein–RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host–pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.

Synthesis of phosphorus mono- and bicycles by catalytic ring-closing metathesis

A versatile route of synthesis of phosphorus oxide and phosphorus borane templates starting from the bifunctional phosphorylating agent bis(diisopropylamino)ethynylphosphine is presented. Ring-closing enyne metathesis using 4,5-dihydro-imidazol-2-ylidene ruthenium benzylidene complex 3 on these types of substrates led to the formation of mono- and bicyclic phosphorus heterocycles.

TLR Ligand–Peptide Conjugate Vaccines: Toward Clinical Application

Approaches to treat cancer with therapeutic vaccination have made significant progress. In order to induce efficient antitumor immunity, a vaccine should target and activate antigen-presenting cells, such as the dendritic cell, while delivering the tumor-derived antigen of choice. Conjugates of synthetic peptides and ligands of pattern-recognition receptors (PRRs) combine these features and, given their synthetic nature, can be produced under GMP conditions. Therefore, conjugation of antigenic peptides to potent PRR ligands is a promising vaccination approach for the treatment of cancer. This review focuses on the different PRR families that can be exploited for the design of conjugates and explores the results obtained so far with PRR ligands conjugated to antigen. The uptake and processing of Toll-like receptor ligand-peptide conjugates are discussed in more detail, as well as future directions that may further enhance the immunogenicity of conjugates.