Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coil cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of Pdu

The Amino Terminus of the Yeast F_1-ATPase β-Subunit Precursor Functions as a Mitochondrial Import Signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Oral Protein Therapy for the Future - Transport of Glycolipid-Modified Proteins: Vision or Fiction?

The reliable and early diagnosis of common complex multifactorial diseases depends on the individual determination of all (or as many as possible) polymorphisms of each susceptibility gene together with amount and type of the corresponding gene products and their downstream effects, including the synthesis and flux of metabolites and regulation of signalling processes. In addition, this system's biology-driven personalized diagnosis must be accompanied by options for personalized reliable and early therapy. In the midterm, the direct substitution or inhibition of the proteins encoded by the corresponding defective gene products of the susceptibility genes exerting lower or higher activity by administration of the `normal' proteins or inhibitory antibodies, respectively, seems to be most promising. The critical hurdle of oral bioavailability as well as transport into the cytoplasm of the target cells, if required, could be overcome by therapeutic proteins with carboxy-terminal modification by glycosylphosphatidylinositol (GPI). This may be deduced from recent experiments with rat adipocytes. Here this membrane-anchoring glycolipid structure induces the sequential transport of proteins from special regions of the plasma membrane via the surface of intracellular lipid droplets to special membrane vesicles, which are finally released from the adipocytes together with the associated GPI proteins. It remains to be studied whether similar molecular mechanisms operate in intestinal epithelial cells and may enable the transport of GPI proteins from the intestinal lumen into the blood stream. If so, modification of proteins encoded by (combinations of) susceptibility genes with GPI could significantly facilitate the personalized therapy of common diseases on the basis of `inborn' safety, efficacy, rapid realization and oral application. Copyright (C) 2010 S. Karger AG, Base

The endoplasmic reticulum in plant immunity and cell death

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants

Callose homeostasis at plasmodesmata: molecular regulators and developmental relevance

Plasmodesmata are membrane-lined channels that are located in the plant cell wall and that physically interconnect the cytoplasm and the endoplasmic reticulum (ER) of adjacent cells. Operating as controllable gates, plasmodesmata regulate the symplastic trafficking of micro- and macromolecules, such as endogenous proteins [transcription factors (TFs)] and RNA-based signals (mRNA, siRNA, etc.), hence mediating direct cell-to-cell communication and long distance signaling. Besides this physiological role, plasmodesmata also form gateways through which viral genomes can pass, largely facilitating the pernicious spread of viral infections. Plasmodesmatal trafficking is either passive (e.g., diffusion) or active and responses both to developmental and environmental stimuli. In general, plasmodesmatal conductivity is regulated by the controlled build-up of callose at the plasmodesmatal neck, largely mediated by the antagonistic action of callose synthases (CalSs) and beta-1,3-glucanases. Here, in this theory and hypothesis paper, we outline the importance of callose metabolism in PD SEL control, and highlight the main molecular factors involved. In addition, we also review other proteins that regulate symplastic PD transport, both in a developmental and stress-responsive framework, and discuss on their putative role in the modulation of PD callose turn-over. Finally, we hypothesize on the role of structural sterols in the regulation of (PD) callose deposition and outline putative mechanisms by which this regulation may occur

Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus

[EN] Replication of all positive RNA viruses occurs in association with intracellular membranes. In many cases, the mechanism of membrane targeting is unknown and there appears to be no correlation between virus phylogeny and the membrane systems recruited for replication. Pelargonium flower break virus (PFBV, genus Carmovirus, family Tombusviridae) encodes two proteins, p27 and its read-through product p86 (the viral RNA dependent-RNA polymerase), that are essential for replication. Recent reports with other members of the family Tombusviridae have shown that the smaller replicase protein is targeted to specific intracellular membranes and it is assumed to determine the subcellular localization of the replication complex. Using in vivo expression of green fluorescent protein (GFP) fusions in plant and yeast cells, we show here that PFBV p27 localizes in mitochondria. The same localization pattern was found for p86 that contains the p27 sequence at its N-terminus. Cellular fractionation of p27GFP-expressing cells confirmed the confocal microscopy observations and biochemical treatments suggested a tight association of the protein to membranes. Analysis of deletion mutants allowed identification of two regions required for targeting of p27 to mitochondria. These regions mapped toward the N- and C-terminus of the protein, respectively, and could function independently though with distinct efficiency. In an attempt to search for putative cellular factors involved in p27 localization, the subcellular distribution of the protein was checked in a selected series of knockout yeast strains and the outcome of this approach is discussed. (C) 2011 Elsevier B.V. All rights reserved.This research was supported by grant BFU2006-11230 and BFU2009-11699 from MEC and MICINN (Spain), respectively, and by grants ACOM/2006/210 and ACOMP/2009/040 (Generalitat Valenciana, GV) to C.H. S.M.-T. was the recipient of a predoctoral fellowship from GV and of a predoctoral contract from MEC.Martínez Turiño, S.; Hernandez Fort, C. (2012). Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus. Virus Research. 163(2):580-591. https://doi.org/10.1016/j.virusres.2011.12.011S580591163

MIR376A is a regulator of starvation-induced autophagy

Background: Autophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration. Methods: Over-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3’ UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR. Results: Here, we demonstrated that, a microRNA (miRNA) from the DlkI/Gtl2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh-7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3’ UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role. Conclusions: Our findings underline the importance of miRNAs encoded by the DlkI/Gtl2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy

Peroxissomas e infeções virais: para além da defesa antiviral

Peroxisomes are multifunctional intracellular organelles, crucial for different physiological and pathological processes. Recently, MAVS (mitochondrial antiviral signaling), the mitochondrial adaptor protein essential for the RLR (retinoic acid inducible gene I-like receptors)-mediated antiviral defense was identified at peroxisomes. Upon infection, viral RNA is recognized by RLRs which induce a signaling cascade that initiates with MAVS activation at both mitochondria and peroxisomes. This culminates with the production of antiviral effectors, such as type I IFNs (interferons) and ISGs (IFN-stimulated genes) that prevent viral replication and dissemination. It has been demonstrated that peroxisomal and mitochondrial MAVS act together in a complementing manner: peroxisomal MAVS induces a rapid but short-term response, while mitochondrial MAVS leads to a delayed but long-lasting antiviral response. The aim of this work was to understand the importance of peroxisomes in the cellular antiviral defense and in viral infections. The results presented in this thesis prove that HCV (hepatitis C virus) NS3-4A and HCMV (human cytomegalovirus) vMIA target peroxisomes to inhibit peroxisomal MAVSdependent antiviral signaling, impairing the production of ISGs. We show that NS3-4A inhibits peroxisomal-dependent signaling through the cleavage of peroxisomal MAVS cytosolic domain. We also show that vMIA interacts with peroxisomal MAVS, inhibiting its oligomerization and impairing the downstream signaling. Additionally, vMIA induces peroxisomal fragmentation, which we prove to be independent of the vMIA-mediated peroxisomal MAVS inhibition. Moreover, we show that vMIA is dependent of MFF, an adaptor protein of peroxisomal fission machinery, and we demonstrate that MFF mediates the interaction between vMIA and peroxisomal MAVS. Finally, we present an interactome of protein-protein interactions between human viruses and peroxisomes, revealing that distinct viruses target peroxisomal proteins. A detailed analysis of the identified interaction revealed that lipid metabolism may be the main peroxisomal function exploited by viruses, possibly to enhance viral infection, or for cellular host defense. Altogether, these results enforce the role of peroxisomes as platforms for RLR signaling and, moreover, suggest that their importance for viral infection may go beyond the antiviral defense. Further studies are proposed to better disclose the role of peroxisomes in viral infection, which can ultimately lead to the discovery of novel targets for the development antiviral therapeutics.Os peroxissomas são organelos intracelulares multifuncionais, cruciais para diferentes processos fisiológicos e patológicos. Recentemente, a MAVS, proteína adaptadora mitocondrial essencial para a defesa antiviral mediada pelos recetores RLR, foi identificada nos peroxissomas. Após infeção, o ácido ribonucleico viral é reconhecido pelos recetores RLRs que induzem uma cascata de sinalização que se inicia com a ativação da MAVS, tanto nas mitocôndrias como nos peroxissomas, culminando com a produção de efetores antivirais, tais como interferões do tipo I e genes induzidos pelos interferões (ISGs), que impedem a replicação e disseminação viral. Foi demonstrado que as MAVS peroxissomais e mitocondriais atuam de forma complementar: a MAVS peroxissomal induz uma resposta rápida, mas de curto prazo, enquanto que a MAVS mitocondrial leva a uma resposta antiviral tardia, porém duradoura. O objetivo deste trabalho foi compreender a importância dos peroxissomas na defesa antiviral celular e nas infeções virais. Os resultados apresentados nesta tese provam que a NS3-4A do vírus da hepatite C (HCV) e a vMIA do citomegalovírus humano (HCMV) exploram os peroxissomas para inibir a sinalização antiviral dependente da MAVS, impedindo a produção dos ISGs. Mostramos que a NS3-4A inibe a sinalização dependente dos peroxissomas através da clivagem do domínio citosólico da MAVS e também mostramos que a vMIA interage com a MAVS peroxissomal, inibindo a sua oligomerização e impedindo a sinalização a jusante. Para além disso, a vMIA induz a fragmentação peroxissomal, que provamos ser independente da inibição da sinalização antiviral. Mostramos também que a vMIA é dependente da MFF, uma proteína adaptadora da fissão peroxissomal, e demonstramos que a MFF medeia a interação entre a vMIA e a MAVS peroxissomal. Finalmente, apresentamos um interatoma das interações proteína-proteína entre vírus humanos e peroxissomas, revelando que vírus distintos interagem com diferentes proteínas peroxissomais. Uma análise detalhada das interações identificadas revelou que o metabolismo lipídico pode ser a principal função peroxissomal explorada pelos vírus, possivelmente para aumentar a infeção viral, ou para a defesa do hospedeiro celular. Em conjunto, estes resultados reforçam o papel dos peroxissomas como plataformas para a sinalização dos RLRs e, além disso, sugerem que a sua importância para a infeção viral pode ir além da defesa antiviral. Novos estudos são propostos para compreender melhor o papel dos peroxissomas na infeção viral, o que pode levar à descoberta de novos alvos para o desenvolvimento de terapias antivirais.Programa Doutoral em Biomedicin