Cell type-specific labeling of newly synthesized proteins by puromycin inactivation.

Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here, we show how a widely used mammalian selection marker, puromycin N-acetyltransferase, can be repurposed for cell-specific metabolic labeling. This approach, which we named puromycin inactivation for cell-selective proteome labeling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing puromycin N-acetyltransferase and detection of translation in other cell types. Using cocultures of neurons and glial cells from the rat brain cortex, we show the application of PICSL for puromycin immunostaining, Western blot, and mass spectrometric identification of nascent proteins. By combining PICSL and OPP-mediated proteomics, cell type-enriched proteins can be identified based on reduced OPP labeling in the cell type of interest.

Revisiting the expression of BDNF and its receptors in mammalian development.

Brain-derived neurotrophic factor (BDNF) promotes the survival and functioning of neurons in the central nervous system and contributes to proper functioning of many non-neural tissues. Although the regulation and role of BDNF have been extensively studied, a rigorous analysis of the expression dynamics of BDNF and its receptors TrkB and p75NTR is lacking. Here, we have analyzed more than 3,600 samples from 18 published RNA sequencing datasets, and used over 17,000 samples from GTEx, and ~ 180 samples from BrainSpan database, to describe the expression of BDNF in the developing mammalian neural and non-neural tissues. We show evolutionarily conserved dynamics and expression patterns of BDNF mRNA and non-conserved alternative 5' exon usage. Finally, we also show increasing BDNF protein levels during murine brain development and BDNF protein expression in several non-neural tissues. In parallel, we describe the spatiotemporal expression pattern of BDNF receptors TrkB and p75NTR in both murines and humans. Collectively, our in-depth analysis of the expression of BDNF and its receptors gives insight into the regulation and signaling of BDNF in the whole organism throughout life.

Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

Deficiency of the Intramembrane Protease SPPL2a Alters Antimycobacterial Cytokine Responses of Dendritic Cells.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartyl intramembrane protease essential for degradation of the invariant chain CD74. In humans, absence of SPPL2a leads to Mendelian susceptibility to mycobacterial disease, which is attributed to a loss of the dendritic cell (DC) subset conventional DC2. In this study, we confirm depletion of conventional DC2 in lymphatic tissues of SPPL2a-/- mice and demonstrate dependence on CD74 using SPPL2a-/- CD74-/- mice. Upon contact with mycobacteria, SPPL2a-/- bone marrow-derived DCs show enhanced secretion of IL-1ß, whereas production of IL-10 and IFN-ß is reduced. These effects correlated with modulated responses upon selective stimulation of the pattern recognition receptors TLR4 and Dectin-1. In SPPL2a-/- bone marrow-derived DCs, Dectin-1 is redistributed to endosomal compartments. Thus, SPPL2a deficiency alters pattern recognition receptor pathways in a CD74-dependent way, shifting the balance from anti- to proinflammatory cytokines in antimycobacterial responses. We propose that in addition to the DC reduction, this altered DC functionality contributes to Mendelian susceptibility to mycobacterial disease upon SPPL2a deficiency.

Physiological functions of SPP/SPPL intramembrane proteases.

Intramembrane proteolysis describes the cleavage of substrate proteins within their hydrophobic transmembrane segments. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3. As presenilin homologues, they employ a similar catalytic mechanism as the well-studied γ-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. The characterisation of SPP/SPPL-deficient mouse models has highlighted a still growing spectrum of biological functions and also promoted the substrate discovery of these proteases. In this review, we will summarise the current hypotheses how phenotypes of these mouse models are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events rather provide specific regulatory switches than unspecific bulk proteolysis. By this means, a plethora of different cell biological pathways is influenced including signal transduction, membrane trafficking and protein glycosylation.

Proteolytic Regulation of the Lectin-Like Oxidized Lipoprotein Receptor LOX-1.

The lectin-like oxidized-LDL (oxLDL) receptor LOX-1, which is broadly expressed in vascular cells, represents a key mediator of endothelial activation and dysfunction in atherosclerotic plaque development. Being a member of the C-type lectin receptor family, LOX-1 can bind different ligands, with oxLDL being the best characterized. LOX-1 mediates oxLDL uptake into vascular cells and by this means can promote foam cell formation. In addition, LOX-1 triggers multiple signaling pathways, which ultimately induce a pro-atherogenic and pro-fibrotic transcriptional program. However, the molecular mechanisms underlying this signal transduction remain incompletely understood. In this regard, proteolysis has recently emerged as a regulatory mechanism of LOX-1 function. Different proteolytic cleavages within the LOX-1 protein can initiate its turnover and control the cellular levels of this receptor. Thereby, cleavage products with individual biological functions and/or medical significance are produced. Ectodomain shedding leads to the release of a soluble form of the receptor (sLOX1) which has been suggested to have diagnostic potential as a biomarker. Removal of the ectodomain leaves behind a membrane-bound N-terminal fragment (NTF), which despite being devoid of the ligand-binding domain is actively involved in signal transduction. Degradation of this LOX-1 NTF, which represents an athero-protective mechanism, critically depends on the aspartyl intramembrane proteases Signal peptide peptidase-like 2a and b (SPPL2a/b). Here, we present an overview of the biology of LOX-1 focusing on how proteolytic cleavages directly modulate the function of this receptor and, what kind of pathophysiological implications this has in cardiovascular disease.

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis.

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.

Identification of Leukotoxin and other vaccine candidate proteins in a Mannheimia haemolytica commercial antigen.

Bovine Respiratory Disease is the most costly disease that affects beef and dairy cattle industry. Its etiology is multifactorial, arising from predisposing environmental stress conditions as well as the action of several different respiratory pathogens. This situation has hindered the development of effective control strategies. Although different type of vaccines are available, many currently marketed vaccines are based on inactivated cultures of the main viral and bacterial agents involved in this pathology. The molecular composition of commercial veterinary vaccines is a critical issue. The present work aims to define at the proteomic level the most relevant valence of a line of commercial respiratory vaccines widely used in Central and South America. Since Mannheimia haemolytica is responsible for most of the disease associated morbid-mortality, we focused on the main proteins secreted by this pathogen, in particular Leukotoxin A, its main virulence factor. By Western blot analysis and mass spectrometry, Leukotoxin A was identified as a major component of M. haemolytica culture supernatants. We also identified other ten M. haemolytica proteins, including outer membrane proteins, periplasmic transmembrane solute transporters and iron binding proteins, which are relevant to achieve protective immunity against the pathogen. This work allowed a detailed molecular characterization of this vaccine component, providing evidence of its quality and efficacy. Furthermore, our results contributed to the identification of several proteins of interest as subunit vaccine candidates.

Silent Polymorphisms: Can the tRNA Population Explain Changes in Protein Properties?

Silent mutations are being intensively studied. We previously showed that the estrogen receptor alpha Ala87's synonymous polymorphism affects its functional properties. Whereas a link has been clearly established between the effect of silent mutations, tRNA abundance and protein folding in prokaryotes, this connection remains controversial in eukaryotic systems. Although a synonymous polymorphism can affect mRNA structure or the interaction with specific ligands, it seems that the relative frequencies of isoacceptor tRNAs could play a key role in the protein-folding process, possibly through modulation of translation kinetics. Conformational changes could be subtle but enough to cause alterations in solubility, proteolysis profiles, functional parameters or intracellular targeting. Interestingly, recent advances describe dramatic changes in the tRNA population associated with proliferation, differentiation or response to chemical, physical or biological stress. In addition, several reports reveal changes in tRNAs' posttranscriptional modifications in different physiological or pathological conditions. In consequence, since changes in the cell state imply quantitative and/or qualitative changes in the tRNA pool, they could increase the likelihood of protein conformational variants, related to a particular codon usage during translation, with consequences of diverse significance. These observations emphasize the importance of genetic code flexibility in the co-translational protein-folding process.

Gene expression changes induced by Trypanosoma cruzi shed microvesicles in mammalian host cells: relevance of tRNA-derived halves.

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

A particular set of small non-coding RNAs is bound to the distinctive Argonaute protein of Trypanosoma cruzi: insights from RNA-interference deficient organisms.

The study of small RNAs and Argonaute proteins in eukaryotes that are deficient in functional RNA interference could provide insights into novel functions of small RNAs. In this study we describe small non-coding RNAs bound to a distinctive Argonaute protein of Trypanosoma cruzi, TcPIWI-tryp. Co-immunoprecipitation of TcPIWI-tryp followed by deep sequencing of isolated RNA identified abundant small RNAs derived from rRNAs and tRNAs. The small RNA repertoire differed from that of the canonical Argonaute in organisms with functional RNA interference, which could indicate novel biological functions for TcPIWI-tryp in T. cruzi and other members of the trypanosomatid clade.

Extracellular vesicles shed by Trypanosoma cruzi are linked to small RNA pathways, life cycle regulation, and susceptibility to infection of mammalian cells.

The protozoan parasite Trypanosoma cruzi has a complex life cycle characterized by intracellular and extracellular forms alternating between invertebrate and mammals. To cope with these changing environments, T. cruzi undergoes rapid changes in gene expression, which are achieved essentially at the posttranscriptional level. At present, expanding families of small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, T. cruzi lacks canonical small RNA pathways. In a recent work, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by a homogeneous population of tRNA-derived small RNAs (tsRNAs). In T. cruzi epimastigotes submitted to nutrient starvation, tsRNAs colocalized with an argonaute protein distinctive of trypanosomatids (TcPIWI-tryp) and were recruited to particular cytoplasmic granules. Using epifluorescence and electronic microscopy, we observed that tsRNAs and the TcPIWI-tryp protein were recruited mainly to reservosomes and other intracellular vesicles including endosome-like vesicles and vesicular structures resembling the Golgi complex. These data suggested that, in T. cruzi, tsRNA biogenesis is probably part of endocytic/exocytic routes. We also demonstrated that epimastigotes submitted to nutrient starvation shed high levels of vesicles to the extracellular medium, which carry small tRNAs and TcPIWI-tryp proteins as cargo. At least a fraction of extracellular vesicle cargo was transferred between parasites and to mammalian susceptible cells. Our data afford experimental evidence, indicating that extracellular vesicles shed by T. cruzi promote not only life cycle transition of epimastigotes to trypomastigote forms but also infection susceptibility of mammalian cells.

Hints of tRNA-Derived Small RNAs Role in RNA Silencing Mechanisms.

With the advent of new and improved high-throughput sequencing technologies in the last few years, a growing number of novel classes of small RNA, other than miRNAs or siRNA, has emerged, which appear as new actors in gene expression regulation. tRNA-derived small RNAs represent one of these novel members that are, surprisingly, among the most conserved class of small RNAs throughout evolution. They could represent the most primitive small RNA pathways from which the well-known canonical RNA silencing pathways reported in higher eukaryotes evolved. This review aims to make a compilation of the most relevant research literature in this field with the purpose of shedding light on the relation of these primitive tRNA-derived molecules with the gene silencing machinery.