Luca Varani

Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release

HMGB1 orchestrates leukocyte recruitment and their induction to secrete inflammatory cytokines by switching between mutually exclusive redox states.

HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4

CXCL12 forms a complex with HMGB1 that binds to the chemokine receptor CXCR4 and increases inflammatory cell migration.

Specificity, cross-reactivity and function of antibodies elicited by Zika virus infection

Characterizing the Zika virus antibody response Given the public health emergency that Zika virus poses, scientists are seeking to understand the Zika-specific immune response. Stettler et al. analyzed 119 monoclonal antibodies isolated from four donors that were infected with Zika virus during the present epidemic, including two individuals that had previously been infected with dengue virus, another member of the flavivirus family. Neutralizing antibodies primarily recognized the envelope protein domain III (EDIII) or quaternary epitopes on the intact virus, and an EDIII-targeted antibody protected mice against lethal infection. Some EDI/II-targeting antibodies cross-reacted with dengue virus in vitro and could enhance disease in dengue-infected mice. Whether dengue and Zika virus antibodies cross-react in humans remains to be tested. Science, this issue p. 823 Cross-reactive antibody responses may pose a risk for disease on secondary infections with Dengue and/or Zika viruses. Zika virus (ZIKV), a mosquito-borne flavivirus with homology to Dengue virus (DENV), has become a public health emergency. By characterizing memory lymphocytes from ZIKV-infected patients, we dissected ZIKV-specific and DENV–cross-reactive immune responses. Antibodies to nonstructural protein 1 (NS1) were largely ZIKV-specific and were used to develop a serological diagnostic tool. In contrast, antibodies against E protein domain I/II (EDI/II) were cross-reactive and, although poorly neutralizing, potently enhanced ZIKV and DENV infection in vitro and lethally enhanced DENV disease in mice. Memory T cells against NS1 or E proteins were poorly cross-reactive, even in donors preexposed to DENV. The most potent neutralizing antibodies were ZIKV-specific and targeted EDIII or quaternary epitopes on infectious virus. An EDIII-specific antibody protected mice from lethal ZIKV infection, illustrating the potential for antibody-based therapy.

The NMR structure of the 38 kDa U1A protein - PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein.

The status of the poly(A) tail at the 3′-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3′-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.

A novel mutation at position +12 in the intron following Exon 10 of the tau gene in familial frontotemporal dementia (FTD‐Kumamoto)

Exonic and intronic mutations in the tau gene cause familial frontotemporal dementia and parkinsonism linked to chromosome 17. Here, we describe a new mutation, consisting of a C‐to‐T transition at position +12 of the intron following exon 10 of the tau gene in the Kumamoto pedigree, showing frontotemporal dementia. The mutation caused a marked reduction in melting temperature of the tau exon 10–splicing regulatory element RNA and a large increase in exon 10–containing transcripts. Brain tissue from affected individuals showed an abnormal preponderance of exon 10–containing transcripts that was reflected at the protein level by an overproduction of tau isoforms with four microtubule‐binding repeats. Immunostaining revealed the presence of tau aggregates in degenerating neurons and glial cells. Isolated tau filaments had a twisted ribbon‐like morphology and were made of hyperphosphorylated four‐repeat tau isoforms. The additional mutation located close to the splice‐donor site of the intron following exon 10 of the tau gene supports the view that intronic mutations exercize their pathogenic effect by destabilizing RNA secondary structure. Ann Neurol 2000;47:422–429.

Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus

Significance Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe respiratory disease with a high mortality rate. There is no licensed vaccine or antiviral for MERS. Here we isolated for the first time, to our knowledge, a potent MERS-CoV–neutralizing antibody from memory B cells of an infected individual. This antibody binds to a novel site on the viral Spike protein, neutralizes by interfering with the binding to the cellular receptor CD26, and is highly effective both in prophylaxis and in therapy in a relevant mouse model. This antibody can be developed for prophylaxis, for postexposure prophylaxis, or for the treatment of severe MERS-CoV infections. Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previously unidentified coronavirus (CoV), likely transmitted to humans by infected camels. There is no licensed vaccine or antiviral for MERS, therefore new prophylactic and therapeutic strategies to combat human infections are needed. In this study, we describe, for the first time, to our knowledge, the isolation of a potent MERS-CoV–neutralizing antibody from memory B cells of an infected individual. The antibody, named LCA60, binds to a novel site on the spike protein and potently neutralizes infection of multiple MERS-CoV isolates by interfering with the binding to the cellular receptor CD26. Importantly, using mice transduced with adenovirus expressing human CD26 and infected with MERS-CoV, we show that LCA60 can effectively protect in both prophylactic and postexposure settings. This antibody can be used for prophylaxis, for postexposure prophylaxis of individuals at risk, or for the treatment of human cases of MERS-CoV infection. The fact that it took only 4 mo from the initial screening of B cells derived from a convalescent patient for the development of a stable chinese hamster ovary (CHO) cell line producing neutralizing antibodies at more than 5 g/L provides an example of a rapid pathway toward the generation of effective antiviral therapies against emerging viruses.

Gold Nanoparticles Downregulate Interleukin‐1β‐Induced Pro‐Inflammatory Responses

Interleukin 1 beta (IL-1β)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1β-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1β both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1β, thus opening potentially novel options for further therapeutic applications.

Refinement of the structure of protein–RNA complexes by residual dipolar coupling analysis

The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein–RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology.

Solution mapping of t cell receptor docking footprints on peptide-MHC

T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR–pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a “recognition code” between conserved elements of the TCR and MHC genes. Although ≈17 cocrystal structures of unique TCR–pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H–2Ld–QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.

Computational Docking of Antibody-Antigen Complexes, Opportunities and Pitfalls Illustrated by Influenza Hemagglutinin

Antibodies play an increasingly important role in both basic research and the pharmaceutical industry. Since their efficiency depends, in ultimate analysis, on their atomic interactions with an antigen, studying such interactions is important to understand how they function and, in the long run, to design new molecules with desired properties. Computational docking, the process of predicting the conformation of a complex from its separated components, is emerging as a fast and affordable technique for the structural characterization of antibody-antigen complexes. In this manuscript, we first describe the different computational strategies for the modeling of antibodies and docking of their complexes, and then predict the binding of two antibodies to the stalk region of influenza hemagglutinin, an important pharmaceutical target. The purpose is two-fold: on a general note, we want to illustrate the advantages and pitfalls of computational docking with a practical example, using different approaches and comparing the results to known experimental structures. On a more specific note, we want to assess if docking can be successful in characterizing the binding to the same influenza epitope of other antibodies with unknown structure, which has practical relevance for pharmaceutical and biological research. The paper clearly shows that some of the computational docking predictions can be very accurate, but the algorithm often fails to discriminate them from inaccurate solutions. It is of paramount importance, therefore, to use rapidly obtained experimental data to validate the computational results.