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The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.

TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase's active site, providing a structural framework for the function of TIPRL in PP2A inhibition.

Background: Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins., Results and Discussion: Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins., Conclusions: This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

Transcription activator-like (TAL) effectors from Xanthomonas species pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the carboxyl-terminal domain of the sweet orange (Citrus sinensis) RNA polymerase II (Pol II) and inhibit the activity of CsCYP, a cyclophilin associated with the carboxyl-terminal domain of the citrus RNA Pol II that functions as a negative regulator of cell growth. Here, we show that PthA4 specifically interacted with the sweet orange MAF1 (CsMAF1) protein, an RNA polymerase III (Pol III) repressor that controls ribosome biogenesis and cell growth in yeast (Saccharomyces cerevisiae) and human. CsMAF1 bound the human RNA Pol III and rescued the yeast maf1 mutant by repressing tRNA(His) transcription. The expression of PthA4 in the maf1 mutant slightly restored tRNA(His) synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNA interference plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis, and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and the presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and Pol III to coordinately increase the transcription of host genes involved in ribosome biogenesis and cell proliferation.

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Type 2A serine/threonine phosphatases are part of the PPP subfamily that is formed by PP2A, PP4 and PP6, and participate in a variety of cellular processes including transcription, translation, regulation of the cell cycle, signal transduction and apoptosis. PP2A is found predominantly as a heterotrimer formed by the catalytic subunit (C) and by a regulatory (B, B' or B'') and a scaffolding (A) subunit. Yeast Tap42p and Tip41p are regulators of type 2A phosphatases, playing antagonistic roles in the target of rapamycin signaling pathway. alpha4 and target of rapamycin signaling pathway regulator-like (TIPRL) are the respective mammalian orthologs of Tap42p and Tip41p. alpha4 has been characterized as an essential protein implicated in cell signaling, differentiation and survival; by contrast, the role of mammalian TIPRL is still poorly understood. In this study, a yeast two-hybrid screen revealed that TIPRL interacts with the C-terminal region of the catalytic subunits of PP2A, PP4 and PP6. Tauhe TIPRL-interacting region on the catalytic subunit was mapped to residues 210-309 and does not overlap with the alpha4-binding region, as shown by yeast two-hybrid and pull-down assays using recombinant proteins. TIPRL and alpha4 can bind PP2Ac simultaneously, forming a stable ternary complex. Reverse two-hybrid assays revealed that single amino acid substitutions on TIPRL including D71L, I136T, M196V and D198N can block its interaction with PP2Ac. TIPRL inhibits PP2Ac activity in vitro and forms a rapamycin-insensitive complex with PP2Ac and alpha4 in human cells. These results suggest the existence of a novel PP2A heterotrimer (alpha4:PP2Ac:TIPRL) in mammalian cells.

The yeast Tap42 and mammalian alpha4 proteins belong to a highly conserved family of regulators of the type 2A phosphatases, which participate in the rapamycin-sensitive signaling pathway, connecting nutrient availability to cell growth. The mechanism of regulation involves binding of Tap42 to Sit4 and PPH21/22 in yeast and binding of alpha4 to the catalytic subunits of type 2A-related phosphatases PP2A, PP4 and PP6 in mammals. Both recombinant proteins undergo partial proteolysis, generating stable N-terminal fragments. The full-length proteins and alpha4 C-terminal deletion mutants at amino acids 222 (alpha4Delta222), 236 (alpha4Delta236) and 254 (alpha4Delta254) were expressed in E. coli. alpha4Delta254 undergoes proteolysis, producing a fragment similar to the one generated by full-length alpha4, whereas alpha4Delta222 and alpha4Delta236 are highly stable proteins. alpha4 and Tap42 show alpha-helical circular dichroism spectra, as do their respective N-terminal proteolysis resistant products. The cloned truncated proteins alpha4Delta222 and alpha4Delta236, however, possess a higher content of alpha-helix, indicating that the C-terminal region is less structured, which is consistent with its higher sensitivity to proteolysis. In spite of their higher secondary structure content, alpha4Delta222 and alpha4Delta236 showed thermal unfolding kinetics similar to the full-length alpha4. Based on small angle X-ray scattering (SAXS), the calculated radius of gyration for alpha4 and Tap42 were 41.2 +/- 0.8 A and 42.8 +/- 0.7 A and their maximum dimension approximately 142 A and approximately 147 A, respectively. The radii of gyration for alpha4Delta222 and alpha4Delta236 were 21.6 +/- 0.3 A and 25.7 +/- 0.2 A, respectively. Kratky plots show that all studied proteins show variable degree of compactness. Calculation of model structures based on SAXS data showed that alpha4Delta222 and alpha4Delta236 proteins have globular conformation, whereas alpha4 and Tap42 exhibit elongated shapes.