Adelante / Endavant

Séptimo desafío por la erradicación de la violencia contra las mujeres del Institut Universitari d’Estudis Feministes i de Gènere "Purificación Escribano" de la Universitat Jaume

The Nβ motif of NaTrxh directs secretion as an endoplasmic reticulum transit peptide and variations might result in different cellular targeting.

Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by the signal recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nβ, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues in plant proteins-a negative residue in position 8 is suggested in animal proteins-to maintain the Nβ motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nβ motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

Summary: Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach

The anaerobic catabolism of aromatic compounds: a genetic and genomic view

63 páginas, 21 figuras, 3 tablas -- PAGS nros. 71-133Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approachWork in our laboratory was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (BIO2003-01482, VEM2003-20075-C02-02, GEN2006-27750-C5-3-E, BIO2006-05957, and CSD2007-00005) and the Comunidad Autónoma de Madrid (P-AMB-259-0505)Peer reviewe

The Nβ “semi-conventional” secretion pathway differs from the conventional and unconventional pathways.

In the conventional secretion pathway (CSP), during translation of a protein with a signal peptide (SP), the SP is recognized by SRP, resulting in the translocation of the mRNA-ribosome-nascent protein to the endoplasmic reticulum lumen, where the SP is cleaved, and translation is completed. The unconventional secretion pathways (USP) comprise: Golgi apparatus bypass (1), where the protein is directly secreted from the ER; ER-independent routes, where proteins may follow secretion after Golgi incorporation (2), through incorporation in multivesicular bodies (3), or through alternative direct secretion pathways (4). In the case of Nβ motif-containing proteins, such as NaTrxh, these are translated in the cytosol and, in an SRP-independent manner, are translocated to the ER to then progress to secretion via the CSP elements (ER, Golgi and secretory vesicles). Figure created using Biorender.</p

The GFP-Nβ fusion protein uses the ER for its secretion.

(A) Schematic representation of the GFP-Nβ construct with the ER retention signal KDEL [GFP-Nβ(KDEL)]. (B) Transient expression assay in onion epidermal cells of the GFP-Nβ(KDEL) protein. Nuclei were stained with propidium iodide before observation (magenta fluorescence). Left panels: GFP fluorescence; middle panels: propidium iodide fluorescence; right panels: GFP and nucleus-labelled merged image (upper) and bright field, GFP and nucleus-labelled merged image (lower). Cyt: cytoplasm; CW: cell wall; Nu: nucleus. Scale bar: 50 μm.</p

The first three Nβ motif residues do not contribute to its secretion signal function.

(A) Schematic representation of the two types of constructs generated to assess the size of the functional Nβ motif. In one, deletions were made from NaTrxh fused to GFP (left) and the other from the Nβ motif directly fused to GFP (right). The Nα motif is displayed in orange, the Nβ in red, the core of NaTrxh in blue, the C-motif in cyan and GFP in green. Light grey represents the deleted residues in both types of constructs; for the Nβ motif directly fused to GFP (right), in dark grey are indicated the initial methionine and the linker sequence between the insert and the GFP sequence. (B) Transient expression assays in onion epidermal cells and observed under confocal microscopy for GFP fluorescence to determine the localisation of NaTrxh-GFP, Nβ-GFP, NaTrxhΔNα(+3)-GFP, Nβ(-3)-GFP, NaTrxhΔNα(+6)-GFP, Nβ(-6)-GFP. Left panels: GFP fluorescence; right panels: merged image of bright field plus GFP fluorescence. Cells were pre-incubated with 1 M NaCl for plasmolysis. Cyt: cytoplasm; CW: cell wall. Scale bar: 50 μm.</p

Positions 5, 8 and 9 within the Nβ motif differ between secretory and cytosolic proteins.

(A) Distribution of the protein sequences among the categories generated based on the UniProtKB database regarding cellular localisation: A category (2.63%); B category (25.66%); C category (2.63%); D category (69.08%). (B) Logos of the sequences found in A category (upper) and C category proteins (down). (C) Transient expression assays in onion epidermal cells (as in Fig 2) of the different Nβ variants fused to GFP. Cyt: cytoplasm; CW: cell wall. Scale bar: 50 μm.</p

The Nβ motif sequence is found in proteins from all taxa.

(A) Schematic representation of the primary structure of NaTrxh, where the Nα motif (orange) comprises from Met-1 (M1) to Ala-16 (A16); the Nβ motif (red), which sequence is shown (Ala-17 to Pro-27) [colour code refers to the charge of each amino acid (green: neutral; black: hydrophobic; red: negative)]; the active site (grey) contains the typical WCGPC motif; the C-terminal extension is shown in cyan and comprises from Glu-136 (E136) to Gln-152 (Q152). (B) Distribution of the eukaryotic proteins (304 sequences) that contain the Nβ motif or a similar one. (C) Consensus sequence (featured as logos) of all the Nβ motifs found in eukaryotic proteins. Colour code as in (A).</p

Sequences raised from the BLASTP analysis.

Retrieved sequences from the BLASTP analysis with an identical or similar Nβ motif sequence. Protein sequences are arranged in rows with the information of each one of them in the columns. Column A: assigned number of the proteins, organised according to the taxon each one corresponds to. Column B: GenBank accession code; Column C: name of the protein and the species where it is from; Columns D and E: query coverage and identity percentages with Nβ motif, respectively; Column F: annotated functions; Column G and H: species and taxon where each protein is from, respectively; Column I: amino acid position of the Nβ motif in the primary structure; Column J: position where the Nβ motif locates within the primary structure (P1, P2, P3 or P4, according to the analysis described in the main text); Column K: size of the primary structure; Column L: cellular localisation. EC: extracellular, Cyt: cytoplasmic, NA: data not available; Column M: score regarding the cellular localisation according to the UniProtKB database, where 5 is the highest and 1 the lowest; Column N: score resulted from SignalP 6.0 to predict the presence of a signal peptide, where 1 is a high probability of the presence of a signal peptide; Column O: organelle or cellular region to which the protein is associated (NA: data not available). The colour code represents the different categories generated in this analysis (described in the main text): green (A category), yellow (B category), red (C category) and gray (D category). (XLSX)</p