Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer.

Covalent amino acid modification significantly expands protein functional capability in regulating biological processes. Tyrosine residues can undergo phosphorylation, sulfation, adenylation, halogenation, and nitration. These posttranslational modifications (PTMs) result from the actions of specific enzymes: tyrosine kinases, tyrosyl-protein sulfotransferase(s), adenylate transferase(s), oxidoreductases, peroxidases, and metal-heme containing proteins. Whereas phosphorylation, sulfation, and adenylation modify the hydroxyl group of tyrosine, tyrosine halogenation and nitration target the adjacent carbon residues. Because aberrant tyrosine nitration has been associated with human disorders and with animal models of disease, we have created an updated and curated database of 908 human nitrated proteins. We have also analyzed this new resource to provide insight into the role of tyrosine nitration in cancer biology, an area that has not previously been considered in detail. Unexpectedly, we have found that 879 of the 1971 known sites of tyrosine nitration are also sites of phosphorylation suggesting an extensive role for nitration in cell signaling. Overall, the review offers several forward-looking opportunities for future research and new perspectives for understanding the role of tyrosine nitration in cancer biology.

Congenital diarrhea in a newborn infant: A case report.

BACKGROUND: Microvillus inclusion disease (MVID) is a rare autosomal recessive cause of severe congenital diarrhea with significant morbidity and mortality. Definitive treatment involves bowel transplant. The diagnosis of this condition can be challenging and a few genetic panels are available for the identification of the most common mutations. We present the case of an infant with MVID due to a mutation not reported in the literature before. CASE SUMMARY: We report the case of an infant transferred to our institution with severe diarrhea of unknown etiology, failure to thrive, and significant metabolic derangements. An extensive work-up including stool studies for common gastrointestinal pathogens, abdominal ultrasound, esophagogastroduodenoscopy with duodenal biopsy and flexible sigmoidoscopy failed to reveal a diagnosis. Multiple dietary and formula regimens were introduced but all resulted in voluminous diarrhea. She remained on total parenteral nutrition (TPN) for the duration of her hospital stay. Genetic testing was done and she was subsequently found to have a novel mutation in the MYO5B gene [homozygous mutation for MYO5B c.1462del, p. (Ile488Leufs*93)] giving us the diagnosis of MVID. She remains on TPN while awaiting bowel transplant at the time of the compilation of this case report. CONCLUSION: We report a novel mutation involved in MVID and highlight the importance of considering this disease when faced with a newborn presenting with life threatening diarrhea. At the time of this publication, 232 allelic variations of this gene (MIM#606540) exist in National Center for Biotechnology Information's database. Our patient's mutation has not been reported in literature as a cause of MVID.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.

Cross-neutralization of influenza A viruses mediated by a single antibody loop.

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Protection from the 2009 H1N1 pandemic influenza by an antibody from combinatorial survivor-based libraries.

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 "Swine" H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.

Surrobodies with functional tails.

Surrobodies(2) are a unique type of binding protein based on the pre-B-cell receptor (pre-BCR). The pre-BCR is transiently expressed during development of the antibody repertoire. Unlike heterotetrameric canonical antibodies that are composed of identical pairs of heavy and light chains, the pre-BCR is a heterohexameric complex composed of identical pairs of heavy chains that are each paired with a two-subunit surrogate light chain (SLC). The SLC contains nonimmunoglobulin-like peptide extensions on each of the two SLC components. This arrangement provides unique opportunities for protein engineering by functional derivatization of these nonimmunoglobulin-like tails. Here we report recombinant fusions to these tails with either a fully active cytokine or with single-chain variable fragment (scFv) domains to generate Surrobodies with unique functions or Surrobodies that are bispecific with respect to targeted binding.

Combinatorial surrobody libraries.

A unique type of combinatorial protein libraries has been constructed. These libraries are based on the pre-B cell receptor (pre-BCR). The pre-BCR is a protein that is produced during normal development of the antibody repertoire. Unlike that of canonical antibodies, the pre-BCR subunit is a trimer that is composed of an antibody heavy chain paired with two surrogate light chain (SLC) components. Combinatorial libraries based on these pre-BCR proteins in which diverse heavy chains are paired with a fixed SLC were expressed in mammalian, Escherichia coli, and phagemid systems. These libraries contain members that have nanomolar affinity for antigen. We term this type of antigen-binding protein a "surrobody" to distinguish it from the canonical antibody molecule.

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies.

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

Biochemical and genetic characterization of Yra1p in budding yeast.

Yra1p and its vertebrate homologues bind to the mRNA export factor Mex67p/TAP and are thought to play a role in mRNA export in vivo. To further characterize Yra1p, we used immunoaffinity chromatography to purify endogenous Yra1p complexes. These experiments demonstrated that two importin beta homologues (Kap123p and Pse1p) and the poly A tail-binding proteins Pab1p and Nab2p associate with Yra1p. The other major proteins that associate with Yra1p include proteins involved in mRNA and rRNA processing and the Yra1p-related protein Yra2p. Additional biochemical and genetic experiments suggest a close functional relationship between Yra1p and Yra2p. We generated a temperature-sensitive allele of YRA1 and used it to demonstrate that cells which lack the function of both Yra1p and Yra2p are able to exit a G0 arrest and go through several rounds of cell division before arresting. We also identified high-copy suppressors of the yra1-2 temperature-sensitive growth defect. These include SUB2, a splicing factor important in mRNA export, ULP1, a nuclear cysteine protease localized to the nuclear pore and involved in Smt3p/SUMO processing, and YRA2. Taken together, these results suggest that Yra1p has roles in diverse RNA processing events in addition to a role in mRNA export.