Acclimation strategies of the green alga Chlorella vulgaris to different light regimes revealed by physiological and comparative proteomic analyses.

Acclimation to different light regimes is at the basis of survival for photosynthetic organisms, regardless of their evolutionary origin. Previous research efforts largely focused on acclimation events occurring at the level of the photosynthetic apparatus and often highlighted species-specific mechanisms. Here, we investigated the consequences of acclimation to different irradiances in Chlorella vulgaris, a green alga that is one of the most promising species for industrial application, focusing on both photosynthetic and mitochondrial activities. Moreover, proteomic analysis of cells acclimated to high light (HL) or low light (LL) allowed identification of the main targets of acclimation in terms of differentially expressed proteins. The results obtained demonstrate photosynthetic adaptation to HL versus LL that was only partially consistent with previous findings in Chlamydomonas reinhardtii, a model organism for green algae, but in many cases similar to vascular plant acclimation events. Increased mitochondrial respiration measured in HL-acclimated cells mainly relied on alternative oxidative pathway dissipating the excessive reducing power produced due to enhanced carbon flow. Finally, proteins involved in cell metabolism, intracellular transport, gene expression, and signaling-including a heliorhodopsin homolog-were identified as strongly differentially expressed in HL versus LL, suggesting their key roles in acclimation to different light regimes.

A 6-month inhalation toxicology study in Apoe-/- mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe-/- mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.

Transcription factor proteomics-Tools, applications, and challenges.

Transcription factors (TFs) are a family of DNA-binding proteins whose gene regulatory capabilities are of vital importance in defining the molecular state of a cell. Despite their biological significance, our understanding of TF behavior and function is still limited. This is because we have so far mostly relied on gene expression data to approximate TF protein levels given that the latter information has been notoriously difficult to obtain due to the relatively low expression levels of many TFs. However, significant advances in mass spectrometry technologies combined with the development of sensitive methodologies aimed at detecting TFs are now allowing a transition from a predominantly qualitative to a quantitative protein landscape. Such a paradigm shift is expected to unravel dynamic aspects of TF function, potentially linking TF copy number fluctuations in cells with specific regulatory functions. This in turn may provide novel insights into the regulatory mechanisms underlying a wide range of fundamental and disease-related biological processes. In this review, we will present the latest advances in mass spectrometry-based TF proteomics and describe novel strategies tailored around the quantification of this important family of DNA-binding proteins.

Quantitative isoform-profiling of highly diversified recognition molecules.

Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands.

A mammalian transcription factor-specific peptide repository for targeted proteomics.

Site-specific transcription factors (TFs) play an essential role in mammalian development and function as they are vital for the majority of cellular processes. Despite their biological importance, TF proteomic data is scarce in the literature, likely due to difficulties in detecting peptides as the abundance of TFs in cells tends to be low. In recent years, significant improvements in MS-based technologies in terms of sensitivity and specificity have increased the interest in developing quantitative methodologies specifically targeting relatively lowly abundant proteins such as TFs in mammalian models. Such efforts would be greatly aided by the availability of TF peptide-specific information as such data would not only enable improvements in speed and accuracy of protein identifications, but also ameliorate cross-comparisons of quantitative proteomics data and allow for a more efficient development of targeted proteomics assays. However, to date, no comprehensive TF proteotypic peptide database has been developed. To address this evident lack of TF peptide data in public repositories, we are generating a comprehensive, experimentally derived TF proteotypic peptide spectral library dataset based on in vitro protein expression. Our library currently contains peptide information for 89 TFs and this number is set to increase in the near future. All MS data have been deposited in the ProteomeXchange with identifier PXD001212 (http://proteomecentral.proteomexchange.org/dataset/PXD001212).

A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.

DNA-centered approaches to characterize regulatory protein-DNA interaction complexes.

Gene regulation is mediated by site-specific DNA-binding proteins or transcription factors (TFs), which form protein complexes at regulatory loci either to activate or repress the expression of a target gene. The study of the dynamic properties of these regulatory DNA-binding complexes has so far been dominated by protein-centered methodologies, aiming to characterize the DNA-binding behavior of one specific protein at a time. With the emerging evidence for a role of DNA in allosterically influencing DNA-binding protein complex formation, there is renewed interest in DNA-centered approaches to capture protein complexes on defined regulatory loci and to correlate changes in their composition with alterations in target gene expression. In this review, we present the current state-of-the-art in such DNA-centered approaches and evaluate recent technological improvements in the purification as well as in the identification of regulatory DNA-binding protein complexes within or outside their biological context. Finally, we suggest possible areas of improvement and assess the putative impact of DNA-centered methodologies on the gene regulation field for the forthcoming years.

Imaging mass spectrometry using peptide isoelectric focusing.

Imaging Mass Spectrometry (IMS) has emerged as a powerful technique in the field of proteomics. The use of Immobilized pH Gradient-IsoElectric Focusing (IPG-IEF) is also a new trend, as the first dimension of separation, in shotgun proteomics. We report a combination of these two outstanding technologies. This approach is based on the separation of shotgun-produced peptides by IPG-IEF. The peptides are then transferred by capillarity to a capture membrane, which is then scanned by the mass spectrometer to generate MS images. This high-throughput methodology allows a preview of the sample to be obtained in a single day. We report the application of this new pipeline for differential comparison of the membrane proteome of two different strains of Staphylococcus aureus bacteria in a proof-of-principle experiment.