Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wld s ) gene

Background

The strain of MeCP2^tm1.1Bird mice is a broadly used model for Rett syndrome. Because males carrying the invalidated MeCP2 locus are sterile, this strain has to be maintained in a heterozygous state. All animals therefore have to be genotyped at every generation to discriminate those carrying the invalidated allele (+/- females and y/- males) from those that do not. This is conveniently carried out by PCR on tail genomic DNA but because the primer pairs described initially for this purpose yield very similar size DNA bands on the WT and the KO alleles, this requires to carry out two independent PCR reactions on tail DNA preparations from all animals.

Results

After cloning and sequencing the PCR fragment amplified on the KO allele, we tested several sets of primers that were designed to yield PCR fragments of different sizes on the KO and WT alleles.

Conclusion

We have thus identified a set of three primers that allows for efficient genotyping of the animals by a single PCR reaction. Furthermore, using of this set of primers also resolves a recurrent problem related to the tendency of one of the initial primers to give rise to a non specific band because of its capacity to anneal at both ends of a repeated genomic element which we have identified as MurvyLTR.     

A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition

Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility.     

Functional analysis of the fibrinogen-related scabrous gene from Drosophila melanogaster identifies potential effector and stimulatory protein domains.

The scabrous (sca) gene encodes a secreted dimeric glycoprotein with putative coiled-coil domains N-terminally and a C-terminal region related to the blood clot protein fibrinogen. Homozygous sca mutants have extra bristle organs and rough eyes. We describe a GAL4-based expression system for testing rescue of the sca mutant phenotype by altered SCA proteins and for misexpression. We find that deletion of the fibrinogen-related domain (FReD) greatly decreases SCA function, confirming the importance of this conserved region. SCA function could not be restored by FReDs from human fibrinogen chain genes. However, proteins lacking any FReD still showed some function in both rescue and misexpression experiments, suggesting that putative effector-binding regions lie outside this domain. Consistent with this, proteins expressing only the FReD had no rescuing activity but were recessive negative; i.e., they enhanced the phenotype of sca mutations but had no phenotype in the presence of a wild-type sca allele. This suggests that the FReD contributes to SCA function by binding to other components of the bristle determination pathway, increasing the activity of the linked N-terminal region.     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

Proteomic analysis identifies dysfunction in cellular transport, energy, and protein metabolism in different brain regions of atypical frontotemporal lobar degeneration

Frontotemporal lobar degeneration (FTLD) is an umbrella term for a heterogeneous group of young-onset dementias of uncertain prevalence and incidence worldwide. Atypical cases of FTLD with fused in sarcoma inclusions (aFTLD-U) have been described recently, but their molecular characterization is still due. Using shotgun mass spectrometry, we identified a total of 107 differentially expressed proteins in the prefrontal cortex, cerebellum and occipital lobe from aFTLD-U patients compared to controls. These proteins are involved in a range of biological pathways such as cellular transport in the prefrontal cortex, energy metabolism in the cerebellum, and protein metabolism in the occipital lobe. In addition, they were validated by selective reaction monitoring (SRM). Comparison of the aFTLD-U proteomic findings with similar studies of Alzheimer's disease and schizophrenia led to identification of proteins that may be related to dementias and psychoses, respectively. Further studies of aFTLD-U and other FTLD subtypes are warranted, although this will require intensive biobanking efforts.     

Differential protein expression in the corpus callosum (splenium) of human alcoholics: a proteomics study

It is widely accepted that the chronic use of alcohol induces metabolic abnormalities and neuronal damage in the brain, which can lead to cognitive dysfunction. Neuroimaging studies reveal that alcohol-induced brain damage is region specific and prominent damage has been observed in both gray and white matter of the prefrontal cortex, and a wide range of white matter structures including the corpus callosum. Molecular mechanisms underlying these structural changes are largely unknown. Using proteomics we have analysed the changes in protein expression in the splenium of the corpus callosum in two different alcoholic groups. Protein extracts from splenium of 22 human brains (nine controls, seven uncomplicated alcoholics and six complicated alcoholics with hepatic cirrhosis-designated complicated) were separated using two-dimensional gel electrophorosis. Image analysis revealed that there were significant alterations in protein expression for 25 protein spots in the uncomplicated alcoholic group and 45 in the complicated group compared to control (P<0.05; ANOVA). In a total of 72 spots (identified as 36 proteins), 15 (identified as 14 proteins) spots overlapped between two alcoholic groups. Another 32 protein spots (26 different proteins) were identified only in the complicated alcoholics. It is therefore possible that these 26 proteins in the complicated group are likely to be the results of hepatic compromise. When compared with our previous data of white matter from the prefrontal cortex in alcoholics, large numbers of identified proteins in the splenium are different. This suggests that there may be different mechanisms causing alcohol-induced brain damage in different regions of the white matter. Our data also indicate the importance of other pathways including oxidative stress, lipid peroxidation and apoptosis as potential causes of alcohol-induced brain damage.     

Differential effect of 2-hydroxyoleic acid enantiomers on protein (sphingomyelin synthase) and lipid (membrane) targets

The complex dual mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent anti-tumor compound used in membrane lipid therapy (MLT), has yet to be fully elucidated. It has been demonstrated that 2OHOA increases the sphingomyelin (SM) cell content via SM synthase (SGMS) activation. Its presence in membranes provokes changes in the membrane lipid structure that induce the translocation of PKC to the membrane and the subsequent overexpression of CDK inhibitor proteins (e.g., p21^Cip1). In addition, 2OHOA also induces the translocation of Ras to the cytoplasm, provoking the silencing of MAPK and its related pathways. These two differential modes of action are triggered by the interactions of 2OHOA with either lipids or proteins. To investigate the molecular basis of the different interactions of 2OHOA with membrane lipids and proteins, we synthesized the R and S enantiomers of this compound. A molecular dynamics study indicated that both enantiomers interact similarly with lipid bilayers, which was further confirmed by X-ray diffraction studies. By contrast, only the S enantiomer was able to activate SMS in human glioma U118 cells. Moreover, the anti-tumor efficacy of the S enantiomer was greater than that of the R enantiomer, as the former can act through both MLT mechanisms. The present study provides additional information on this novel therapeutic approach and on the magnitude of the therapeutic effects of type-1 and type-2 MLT approaches. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Chronic morphine alters the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis

Opiates produce significant and persistent changes in synaptic transmission; knowledge of the proteins involved in these changes may help to understand the molecular mechanisms underlying opiate dependence. Using an integrated quantitative proteomics and systems biology approach, we explored changes in the presynaptic protein profile following a paradigm of chronic morphine administration that leads to the development of dependence. For this, we isolated presynaptic fractions from the striata of rats treated with saline or escalating doses of morphine, and analyzed the proteins in these fractions using differential isotopic labeling. We identified 30 proteins that were significantly altered by morphine and integrated them into a protein-protein interaction (PPI) network representing potential morphine-regulated protein complexes. Graph theory-based analysis of this network revealed clusters of densely connected and functionally related morphine-regulated clusters of proteins. One of the clusters contained molecular chaperones thought to be involved in regulation of neurotransmission. Within this cluster, cysteine-string protein (CSP) and the heat shock protein Hsc70 were downregulated by morphine. Interestingly, Hsp90, a heat shock protein that normally interacts with CSP and Hsc70, was upregulated by morphine. Moreover, treatment with the selective Hsp90 inhibitor, geldanamycin, decreased the somatic signs of naloxone-precipitated morphine withdrawal, suggesting that Hsp90 upregulation at the presynapse plays a role in the expression of morphine dependence. Thus, integration of proteomics, network analysis, and behavioral studies has provided a greater understanding of morphine-induced alterations in synaptic composition, and identified a potential novel therapeutic target for opiate dependence.     

Differential regulation of host cellular genes by HIV-1 viral protein R (Vpr): cDNA microarray analysis using isogenic virus

HIV-1 Vpr is a protein with multiple functions. It has been suggested that such pleiotropic effects by a viral protein may be mediated by its association with viral and cellular proteins or through modulation of expression of specific cellular genes. To address this, we used cDNA microarray techniques to analyze the regulation of a panel of host cellular genes by HIV-1 Vpr using isogenic HIV-1 either with or without Vpr expression. Results indicate that Vpr downregulated the expression of genes involved in cell cycle/proliferation regulation, DNA repair, tumor antigens, and immune activation factors, and upregulated many ribosomal and structural proteins. These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule.     

Differential proteomic analysis of the mouse retina: the induction of crystallin proteins by retinal degeneration in the rd1 mouse

We have applied proteomic analysis to the degeneration of photoreceptors. In the rd1 mouse, a recessive mutation in the PDE6B gene leads to rapid loss of rods through apoptosis. By 5 wk postnatal, virtually all rod photoreceptors have degenerated, leaving one row of cones that degenerates secondarily. In order to assess comparative protein expression, proteins extracted from whole retina were resolved on a two-dimensional gel and identified by mass spectrometry combined with database screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry coupled to peptide mass fingerprinting was sufficient to identify most of the proteins, the remaining being identified with additional sequence information obtained by nano-electrospray ionization tandem mass spectrometry or liquid chromatography tandem mass spectrometry. The study revealed 212 spots, grouped into 109 different proteins. Differential analysis showed loss of proteins involved in the rod-specific phototransduction cascade, as well as induction of proteins from the crystallin family, in response to retinal degeneration. Identification of such pathways may contribute to new therapeutic approaches.     

Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery

Influenza A virus NS1 protein (NS1A protein) via its effector domain targets the poly(A)-binding protein II (PABII) of the cellular 3'-end processing machinery. In vitro the NS1A protein binds the PABII protein, and in vivo causes PABII protein molecules to relocalize from nuclear speckles to a uniform distribution throughout the nucleoplasm. In vitro the NS1A protein inhibits the ability of PABII to stimulate the processive synthesis of long poly(A) tails catalyzed by poly(A) polymerase (PAP). Such inhibition also occurs in vivo in influenza virus-infected cells, where the NS1A protein via its effector domain causes the nuclear accumulation of cellular pre-mRNAs which contain short ( approximately 12 nucleotide) poly(A) tails. Consequently, although the NS1A protein also binds the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF), 3' cleavage of some cellular pre-mRNAs still occurs in virus-infected cells, followed by the PAP-catalyzed addition of short poly(A) tails. Subsequent elongation of these short poly(A) tails is blocked because the NS1A protein inhibits PABII function. Nuclear-cytoplasmic shuttling of PABII, an activity implicating this protein in the nuclear export of cellular mRNAs, is also inhibited by the NS1A protein. In vitro assays suggest that the 30 kDa CPSF and PABII proteins bind to non-overlapping regions of the NS1A protein effector domain and indicate that these two 3' processing proteins also directly bind to each other.     

Thiol redox proteomics identifies differential targets of cytosolic and mitochondrial glutaredoxin-2 isoforms in Saccharomyces cerevisiae. Reversible S-glutathionylation of DHBP synthase (RIB3)

Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms.     

Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.     

Receptor interacting protein is ubiquitinated by cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) in vitro

Receptor interacting protein (RIP) is recruited to tumor necrosis factor-alpha receptor 1 (TNFR1) complex upon stimulation and plays a crucial role in the receptor-mediated NF-kappaB activation. Among the components of the TNFR1 complex are proteins that possess ubiquitin-protein isopeptide ligase (E3) activities, such as TNFR1-associated factor 2 (TRAF2), cellular inhibitor of apoptosis proteins (c-IAPs) namely, c-IAP1 and c-IAP2. Here, we showed that ectopically expressed RIP is ubiquitinated, and either the intermediate or death domain of RIP is required for this modification. Expression of c-IAP1 and c-IAP2 decreased the steady-state level of RIP, which was blocked by inhibition of the 26S proteasome. RIP degradation requires intact c-IAP2 containing the RING domain. Our in vitro ubiquitination assay revealed that while TRAF2 had no effect, both c-IAP1 and c-IAP2-mediated RIP ubiquitination with similar efficiency, indicating that c-IAPs can function as E3 toward RIP.