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Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: “In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?” In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells (‘SILAC’) coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter suggests that long-term regulatory processes may contribute to actomyosin-dependent trafficking of the water channel aquaporin-2. The results provide impetus for increased focus on translational regulation and regulation of protein degradation in physiological control in mammalian epithelial cells.
Quantitative Protein and mRNA Profiling Shows Selective Post-Transcriptional Control of Protein Expression by Vasopressin in Kidney Cells*An external file that holds a picture, illustration, etc. Object name is sbox.jpg
Sookkasem Khositseth,‡ Trairak Pisitkun,‡ Dane H. Slentz,‡ Guanghui Wang,‡ Jason D. Hoffert,‡ Mark A. Knepper,‡ and Ming-Jiun Yu‡§
The protozoan parasite Giardia is a highly prevalent intestinal pathogen with a wide host range. Data obtained in mice, cattle and humans revealed the importance of IL-17A in the development of a protective immune response against Giardia. The aim of this study was to further unravel the protective effector mechanisms triggered by IL-17A following G. muris infection in mice, by an RNA-sequencing approach. C57BL/6 WT and C57BL/6 IL-17RA KO mice were orally infected with G. muris cysts. Three weeks post infection, intestinal tissue samples were collected for RNA-sequencing, with samples from uninfected C57BL/6 WT and C57BL/6 IL-17RA KO animals serving as negative controls. Differential expression analysis showed that G. muris infection evoked the transcriptional upregulation of a wide array of genes, mainly in animals with competent IL-17RA signaling. IL-17RA signaling induced the production of various antimicrobial peptides, such as angiogenin 4 and α- and β-defensins and regulated complement activation through mannose-binding lectin 2. The expression of the receptor that regulates the secretion of IgA into the intestinal lumen, the polymeric immunoglobulin receptor, was also dependent on IL-17RA signaling. Interestingly, the transcriptome data showed for the first time the involvement of the circadian clock in the host response following Giardia infection.
Interleukin-17 receptor A (IL-17RA) as a central regulator of the protective immune response against Giardia
Oonagh Paerewijck,1 Brecht Maertens,1 Leentje Dreesen,1 Frederik Van Meulder,1 Iris Peelaers,1 Dariusz Ratman,2 Robert W. Li,3 Erik Lubberts,4 Karolien De Bosscher,2 and Peter Geldhofcorresponding author1
Imbalance of gamma aminobutyric acid (GABA) and related modulators has been implicated as an important factor in the pathogenesis of amyotrophic lateral sclerosis (ALS), which is also known as motor neuron disease (MND). In this context, the role and mechanism of action of gabapentin and baclofen have been extensively investigated, although with conflicting results. This is the first systematic review to assess clinical trials of GABA modulators for the treatment of ALS.
To examine the efficacy of gabapentin, baclofen, or other GABA modulators in delaying the progression of ALS, and to evaluate adverse effects of these interventions.
On 16 August 2016, we searched the Cochrane Neuromuscular Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, CINAHL Plus, AMED, and LILACS. In addition, we checked the bibliographies of the trials found in order to identify any other trials, and contacted trial authors to identify relevant unpublished results or additional clinical trials. On 30 August 2016, we searched two clinical trials registries.
Types of studies: double‐blind randomized controlled trials (RCTs) or quasi‐RCTs
Types of participants: adults with a diagnosis of probable or definite ALS
Types of interventions: gabapentin, baclofen, or other GABA modulators compared with placebo, no treatment, or each other
Primary outcome: survival at one year from study enrollment
Secondary outcomes: individual rate of decline of maximum voluntary isometric contraction (MVIC), expressed as arm megascore; rate of decline of per cent predicted forced vital capacity (FVC); rate of decline of ALS Functional Rating Scale (ALSFRS); health‐related quality of life; survival evaluated by pooling hazards; and adverse events
Data collection and analysis
At least two review authors independently checked titles and abstracts identified by the searches. The review authors obtained and independently analyzed original individual participant data from each included study; additional review authors and the Cochrane Neuromuscular Managing Editor checked the outcome data. Two authors independently assessed the risk of bias in included studies.
We identified two double‐blind RCTs of gabapentin treatment in ALS for inclusion in this review. We found no eligible RCTs of baclofen or other GABA modulators. The selected studies were phase II and phase III trials, which lasted six and nine months, respectively. They were highly comparable because both were comparisons of oral gabapentin and placebo, performed by the same investigators. The trials enrolled 355 participants with ALS: 80 in the gabapentin group and 72 in the placebo group in the first (phase II) trial and 101 in the gabapentin group and 102 in the placebo group in the second (phase III) trial. Neither trial was long enough to report survival at one year, which was our primary outcome. We found little or no difference in estimated one‐year survival between the treated group and the placebo group (78% versus 77%, P = 0.63 by log‐rank test; high‐quality evidence). We also found little or no difference in the rate of decline of MVIC expressed as arm megascore, or rate of FVC decline (high‐quality evidence). One trial investigated monthly decline in the ALSFRS and quality of life measured using the 12‐Item Short Form Survey (SF‐12) and found little or no difference between groups (moderate‐quality evidence). The trials reported similar adverse events. Complaints that were clearly elevated in those taking gabapentin, based on analyses of the combined data, were light‐headedness, drowsiness, and limb swelling (high‐quality evidence). Fatigue and falls occurred more frequently with gabapentin than with placebo in one trial, but when we combined the data for fatigue from both trials, there was no clear difference between the groups. We assessed the overall risk of bias in the included trials as low.
According to high‐quality evidence, gabapentin is not effective in treating ALS. It does not extend survival, slow the rate of decline of muscle strength, respiratory function and, based on moderate‐quality evidence, probably does not improve quality of life or slow monthly decline in the ALSFRS. Other GABA modulators have not been studied in randomized trials.
Gamma aminobutyric acid (GABA) modulators for amyotrophic lateral sclerosis/motor neuron disease
Monitoring Editor: Andrea Diana,corresponding author Rita Pillai, Paolo Bongioanni, Aidan G O'Keeffe, Robert G Miller, Dan H Moore, and Cochrane Neuromuscular Group University of Cagliari, Department of Biomedical Sciences, Citta Universitaria di Monserrato (Cagliari), Monserrato (Cagliari)Italy, 09042 University of Pisa, Neurorehabilitation Unit, Department of Neuroscience, Via Paradisa, 2, PisaItaly, 56100 University College London, Department of Statistical Science, 1‐19 Torrington Place, LondonUK, WC1E 6BT California Pacific Medical Center, Forbes Norris ALS Research Center, 2324 Sacramento Street, Suite 150, San FranciscoUSA, 94115 California Pacific Medical Center, Research Institute, 475 Brannan St Suite 220, San FranciscoUSA, 94107