Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever.

Mayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

The multiple ways Wnt signaling contributes to acute leukemia pathogenesis.

WNT proteins constitute a very conserved family of secreted glycoproteins that act as short-range ligands for signaling with critical roles in hematopoiesis, embryonic development, and tissue homeostasis. These proteins transduce signals via the canonical pathway, which is ß-catenin-mediated and better-characterized, or via more diverse noncanonical pathways that are ß-catenin independent and comprise the planar cell polarity (PCP) pathway and the WNT/Ca++ pathways. Several proteins regulate Wnt signaling through a variety of sophisticated mechanisms. Disorders within the pathway can contribute to various human diseases, and the dysregulation of Wnt pathways by different molecular mechanisms is implicated in the pathogenesis of many types of cancer, including the hematological malignancies. The types of leukemia differ considerably and can be subdivided into chronic, myeloid or lymphocytic, and acute, myeloid or lymphocytic, leukemia, according to the differentiation stage of the predominant cells, the progenitor lineage, the diagnostic age strata, and the specific molecular drivers behind their development. Here, we review the role of Wnt signaling in normal hematopoiesis and discuss in detail the multiple ways canonical Wnt signaling can be dysregulated in acute leukemia, including alterations in gene expression and protein levels, epigenetic regulation, and mutations. Furthermore, we highlight the different impacts of these alterations, considering the distinct forms of the disease, and the therapeutic potential of targeting Wnt signaling.

An Essential Role for ECSIT in Mitochondrial Complex I Assembly and Mitophagy in Macrophages.

ECSIT is a mitochondrial complex I (CI)-associated protein that has been shown to regulate the production of mitochondrial reactive oxygen species (mROS) following engagement of Toll-like receptors (TLRs). We have generated an Ecsit conditional knockout (CKO) mouse strain to study the in vivo role of ECSIT. ECSIT deletion results in profound alteration of macrophage metabolism, leading to a striking shift to reliance on glycolysis, complete disruption of CI activity, and loss of the CI holoenzyme and multiple subassemblies. An increase in constitutive mROS production in ECSIT-deleted macrophages prevents further TLR-induced mROS production. Surprisingly, ECSIT-deleted cells accumulate damaged mitochondria because of defective mitophagy. ECSIT associates with the mitophagy regulator PINK1 and exhibits Parkin-dependent ubiquitination. However, upon ECSIT deletion, we observed increased mitochondrial Parkin without the expected increase in mitophagy. Taken together, these results demonstrate a key role of ECSIT in CI function, mROS production, and mitophagy-dependent mitochondrial quality control.

Interferon regulatory factor 2 binding protein 2 is a new NFAT1 partner and represses its transcriptional activity.

The nuclear factor of activated T cells (NFAT) family of transcription factors is expressed in a wide range of cell types and regulates genes involved in cell cycle, differentiation, and apoptosis. NFAT proteins share two well-conserved regions, the regulatory domain and the DNA binding domain. The N- and C-terminal ends are transactivation sites and show less sequence similarity, whereas their molecular functions remain poorly understood. Here, we identified a transcriptional repressor, interferon regulatory factor 2 binding protein 2 (IRF-2BP2), which specifically interacts with the C-terminal domain of NFAT1 among the NFAT family members. IRF-2BP2 was described as a corepressor by inhibiting both enhancer-activated and basal transcription. Gene reporter assays demonstrated that IRF-2BP2 represses the NFAT1-dependent transactivation of NFAT-responsive promoters. The ectopic expression of IRF-2BP2 in CD4 T cells resulted in decreased interleukin-2 (IL-2) and IL-4 production, supporting a repressive function of IRF-2BP2 for NFAT target genes. Furthermore, NFAT1 and IRF-2BP2 colocalized in the nucleus in activated cells, and the mutation of a newly identified nuclear localization signal in the IRF-2BP2 rendered it cytoplasmic, abolishing its repressive effect on NFAT1 activity. Collectively, our data demonstrate that IRF-2BP2 is a negative regulator of the NFAT1 transcription factor and suggest that NFAT1 repression occurs at the transcriptional level.

Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor.

The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignanttransforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44 degrees C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58 degrees C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein- protein interaction.

The crystal structure of the small GTPase Rab11b reveals critical differences relative to the Rab11a isoform.

Rab GTPases constitute the largest family of small monomeric GTPases, including over 60 members in humans. These GTPases share conserved residues related to nucleotide binding and hydrolysis, and main sequence divergences lie in the carboxyl termini. They cycle between inactive (GDP-bound) and active (GTP-bound) forms and the active site regions, termed Switch I and II, undergo the larger conformational changes between the two states. The Rab11 subfamily members, comprising Rab11a, Rab11b, and Rab25, act in recycling of proteins from the endosomes to the plasma membrane, in transport of molecules from the trans-Golgi network to the plasma membrane and in phagocytosis. In this work, we describe Rab11b-GDP and Rab11b-GppNHp crystal structures solved to 1.55 and 1.95 angstroms resolution, respectively. Although Rab11b shares 90% amino acid identity to Rab11a, its crystal structure shows critical differences relative to previously reported Rab11a structures. Inactive Rab11a formed dimers with unusually ordered Switch regions and missing the magnesium ion at the nucleotide binding site. In this work, inactive Rab11b crystallized as a monomer showing a flexible Switch I and a magnesium ion which is coordinated by four water molecules, the phosphate beta of GDP (beta-P) and the invariant S25. S20 from the P-loop and S42 from the Switch I are associated to GTP hydrolysis rate. In the active structures, S20 interacts with the gamma-P oxygen in Rab11b-GppNHp but does not in Rab11a-GppNHp and the Q70 side chain is found in different positions. In the Rab11a-GTPgammaS structure, S40 is closer to S25 and S42 does not interact with the gamma-P oxygen. These differences indicate that the Rab11 isoforms may possess different GTP hydrolysis rates. In addition, the Switch II of inactive Rab11b presents a 3(10)-helix (residues 69-73) that disappears upon activation. This 3(10)-helix is not found in the Rab11a-GDP structure, which possesses a longer alpha2 helix, spanning from residue 73 to 82 alpha-helix 5.

Spectroscopic characterization of the tumor antigen NY-REN-21 and identification of heterodimer formation with SCAND1.

Human NY-REN-21 is a C2H2 type multi-finger protein, with a SCAN domain in the N-terminal region and a predicted coil central region. It represents a putative ortholog of mouse ZFP38, a transcriptional factor that recognizes a bipartite DNA motif and is unable to form homodimers. As shown in this work, NY-REN-21 contains a SCAN domain able to form homodimers and a central region that behaves as an intrinsically disordered protein. The SCAN domain is found in 71 human proteins and its ability to form homo- and heterodimers widens the number of genes that are regulated by this group of transcription factors. NY-REN-21 interaction with SCAND1 was identified using the yeast two-hybrid system and confirmed using recombinant proteins. SCAND1 is a truncated SCAN box protein, lacking the zinc finger region and the NY-REN-21/SCAND1 heterodimer is asymmetric concerning the DNA binding region. This result indicates that NY-REN-21 can function either as a homodimer or as a heterodimer with SCAND1.