Lafora disease E3-ubiquitin ligase malin is related to TRIM32 at both the phylogenetic and functional level

<p>Abstract</p> <p>Background</p> <p>Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin.</p> <p>Results</p> <p>After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex.</p> <p>Conclusions</p> <p>We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins.</p

A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 5′->3′ Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots

International audienceBackgroundMutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3′,(2′),5′-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta.Principal FindingsA fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 3′-polyadenosine 5′-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background.Conclusions/SignificanceOur results indicate that the 3′,(2′),5′-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs.

ALCAM Regulates Motility, Invasiveness, and Adherens Junction Formation in Uveal Melanoma Cells

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM’s role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves

SUMO-Interacting Motifs of Human TRIM5α are Important for Antiviral Activity

Human TRIM5α potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5α in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5α shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5α revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5α consensus SUMO conjugation site did not affect the antiviral activity of TRIM5α in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5α antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5α restriction of N-MLV. Our data suggest a novel aspect of TRIM5α-mediated restriction, in which the presence of intact SIMs in TRIM5α, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA

Additive Pressures of Elevated Sea Surface Temperatures and Herbicides on Symbiont-Bearing Foraminifera

Elevated ocean temperatures and agrochemical pollution individually threaten inshore coral reefs, but these pressures are likely to occur simultaneously. Experiments were conducted to evaluate the combined effects of elevated temperature and the photosystem II (PSII) inhibiting herbicide diuron on several types of symbiotic algae (diatom, dinoflagellate or rhodophyte) of benthic foraminifera in hospite. Diuron was shown to evoke a direct effect on photosynthetic efficiency (reduced effective PSII quantum yield ΔF/F′m), while elevated temperatures (>30°C, only 2°C above current average summer temperatures) were observed to impact photosynthesis more indirectly by causing reductions in maximum PSII quantum yield (Fv/Fm), interpreted as photodamage. Additionally, elevated temperatures were shown to cause bleaching through loss of chlorophyll a in foraminifera hosting either diatoms or dinoflagellates. A significant linear correlation was found between reduced Fv/Fm and loss of chlorophyll a. In most cases, symbionts within foraminifera proved more sensitive to thermal stress in the presence of diuron (≥1 µg L−1). The mixture toxicity model of Independent Action (IA) described the combined effects of temperature and diuron on the photosystem of species hosting diatoms or dinoflagellates convincingly and in agreement with probabilistic statistics, so a response additive joint action can be assumed. We thus demonstrate that improving water quality can improve resilience of symbiotic phototrophs to projected increases in ocean temperatures. As IA described the observed combined effects from elevated temperature and diuron stress it may therefore be employed for prediction of untested mixtures and for assessing the efficacy of management measures

Engineered Models of Metastasis with Application to Study Cancer Biomechanics

Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

[EN] Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.IRO, VMZM, HHU and PLS were supported by the Mexican National Council of Science and Technology (CONACyT) with a PhD fellowship (210085, 210100, 243380 and 219883, respectively). Work in the SDF laboratory was financed by the CONACyT grants CB-2012-177739, FC-2015-2/1061, and INFR-2015-253504, and NMM by the CONACyT grant CB-2011-165986. SDF, CF and LC acknowledge the support of the European Union FP7-PEOPLE-2009-IRSES project EVOCODE (grant no. 247587) and H2020-MSCARISE-2015 project ExpoSEED (grant no. 691109). SDF also acknowledges the Marine Biological Laboratory (MBL) in Woods Hole for a scholarship for the Gene Regulatory Networks for Development Course 2015 (GERN2015). IE acknowledges the International European Fellowship-METMADS project and the Universita degli Studi di Milano (RTD-A; 2016). Research in the laboratory of MFY was funded by NSF (grant IOS-1121055), NIH (grant 1R01GM112976-01A1) and the Paul D. Saltman Endowed Chair in Science Education (MFY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Reyes Olalde, J.; Zuñiga, V.; Serwatowska, J.; Chávez Montes, R.; Lozano-Sotomayor, P.; Herrera-Ubaldo, H.; Gonzalez Aguilera, K.... (2017). The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium. PLoS Genetics. 13(4):1-31. https://doi.org/10.1371/journal.pgen.1006726S131134Reyes-Olalde, J. I., Zuñiga-Mayo, V. M., Chávez Montes, R. A., Marsch-Martínez, N., & de Folter, S. (2013). Inside the gynoecium: at the carpel margin. Trends in Plant Science, 18(11), 644-655. doi:10.1016/j.tplants.2013.08.002Alvarez-Buylla, E. 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