Protein phosphatase-1α interacts with and dephosphorylates polycystin-1

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.     

Decreased GAD(65) -specific Th1/Tc1 phenotype in children with Type 1 diabetes treated with GAD-alum

Diabet. Med. 29, 1272-1278 (2012) ABSTRACT: Aim The balance between T helper cell subsets is an important regulator of the immune system and is often examined after immune therapies. We aimed to study the immunomodulatory effect of glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) in children with Type?1 diabetes, focusing on chemokines and their receptors. Methods Blood samples were collected from 70 children with Type?1 diabetes included in a phase?II clinical trial with GAD-alum. Expression of CC chemokine receptor?5 (CCR5) and CCR4 was analysed on CD4+ and CD8+ lymphocytes after in vitro stimulation with GAD(65) using flow cytometry, and secretion of the chemokines CCL2, CCL3 and CCL4 was detected in peripheral blood mononuclear cell supernatants with Luminex. Results Expression of Th1-associated CCR5 was down-regulated following antigen challenge, together with an increased CCR4/CCR5 ratio and CCL2 secretion in GAD-alum-treated patients, but not in the placebo group. Conclusion Our results suggest that GAD-alum treatment has induced a favourable immune modulation associated with decreased Th1/Tc1 phenotypes upon antigen re-challenge, which may be of importance for regulating GAD(65) immunity.     

Protein Phosphatase 1 δ is Associated with Focal Adhesions

In all mammalian cells protein phosphatase-1 (PP1) exists in three isoforms, defined as α, γ1 and δ. Immunofluorescence studies with isoform-specific antibodies indicated that δ, but not α or γ1, is enriched at focal adhesions in HeLa cells, fibroblasts, endothelial cells and keratinocytes. This was confirmed also by interference reflection microscopy, which indicated that PP1δ was in areas of tight adhesion of the membrane to the extracellular matrix at sites where the microfilament cytoskeleton is organized. In all the cell types so far considered the PP1δ in focal adhesions represented only a small aliquot of the total PP1δ, which was predominantly localized to the nucleus. The association of PP1δ to focal adhesions was confirmed by the co-immunoprecipitation of PP1δ with the focal adhesion kinase pp125^FAK and with the αv integrin. Comparison between the amount of PP1δ associated with focal adhesion proteins and that of PP1δ recovered in an anti-PP1δ immunoprecipitate confirmed that only a minor amount of the enzyme was associated with the focal adhesions. Since some focal adhesion proteins are phosphorylated on Ser/Thr, it is likely that PP1δ may be involved in the regulation of focal adhesion functions and particularly in the signaling pathway generated by cell-substratum adhesion.     

Monitoring conformational dynamics with solid-state R 1ρ experiments

A new application of solid-state rotating frame (R _1ρ) relaxation experiments to observe conformational dynamics is presented. Studies on a model compound, dimethyl sulfone (DMS), show that R _1ρ relaxation due to reorientation of a chemical shift anisotropy (CSA) tensor undergoing chemical exchange can be used to monitor slow-to-intermediate timescale conformational exchange processes. Control experiments used d ₆ -DMS and alanine to confirm that the technique is monitoring reorientation of the CSA tensor rather than dipolar interactions or methyl group rotation. The application of this method to proteins could represent a new site-specific probe of conformational dynamics.     

Interaction of ZPR1 with Translation Elongation Factor-1α in Proliferating Cells

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1α (eEF-1α). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1α and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH₂-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1α by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1α restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1α contributes to normal cellular proliferation.     

Preferential binding of E. coli with type 1 fimbria to d-mannobiose with the Manα1→2Man structure

Manα1→2Man, Manα1→3Man, Manα1→4Man, and Manα1→6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco? beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1→2Man1→beads, Manα1→3Man1→beads, Manα1→4Man1→beads, and Manα1→6Man1→beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1→2Man disaccharide.     

Are MYO1C and MYO1F associated with hearing loss?

The role of myosins in the pathogenesis of hearing loss is well established: five genes encoding unconventional myosins and two genes encoding nonmuscle conventional myosins have so far been described to be essential for normal auditory function and mutations in these genes associated with hearing impairment. To better understand the role of this gene family we performed a mutational screening on two candidate genes, MYO1C and MYO1F, analyzing hundreds of patients, affected by bilateral sensorineural hearing loss and coming from different European countries. This research activity led to the identification of 6 heterozygous missense mutations in MYO1C and additional 5 heterozygous missense mutations in MYO1F. Homology modelling suggests that some of these mutations could have a potential influence on the structure of the ATP binding site and could probably affect the ATPase activity or the actin binding process of both myosins. This study suggests a role of the above mentioned myosin genes in the pathogenesis of hearing loss.     

−13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1

Lactase gene expression declines with aging (lactase non-persistence) in the majority of humans worldwide. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream (−13907, −13910 and −13915) of the lactase gene in different ethnic populations. In contrast to the −13907*G and −13910*T SNPs, the −13915*G SNP was previously believed not to interact with Oct-1. In the present study, however, Oct-1 is shown to interact with the −13915*G SNP region DNA sequence by EMSAs and gel supershift. In addition, Oct-1 is capable of enhancing promoter activity of a lactase promoter–reporter construct harboring the 13915*G SNP sequence in cell culture. Oct-1 binding to the −13907 to −13915 SNP region therefore remains a candidate interaction involved in lactase persistence.     

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H⁺) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.     

TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions

Background

The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6.

Results

Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site.

Conclusions

Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.     

Does high-mobility-group non-histone protein HMG 1 interact specifically with histone H1 subfractions?

The interaction of the non-histone chromosomal protein HMG (high-mobility group) 1 with histone H1 subfractions was investigated by equilibrium sedimentation and n.m.r. sectroscopy. In contrast with a previous report [Smerdon & Isenberg (1976) Biochemistry 15, 4242--4247], it was found, by using equilibrium-sedimentation analysis, that protein HMG 1 binds to all three histone H1 subfractions CTL1, CTL2, and CTL3, arguing against there being a specific interaction between protein HMG 1 and only two of the subfractions, CTL1 and CTL2. Raising the ionic strength of the solutions prevents binding of protein HMG 1 to total histone H1 and the three subfractions, suggesting that the binding in vitro is simply a non-specific ionic interaction between acidic regions of the non-histone protein and the basic regions of the histone. Protein HMG 1 binds to histone H5 also, supporting this view. The above conclusions are supported by n.m.r. studies of protein HMG 1/histone H1 subfraction mixtures. When the two proteins were mixed, there was little perturbation of the n.m.r. spectra and there was no evidence for specific interaction of protein HMG 1 with any of the subfractions. It therefore remains an open question as to whether protein HMG 1 and histone H1 are complexed together in chromatin.     

Synergism of 1-β-d-arabinofuranosylcytosine with itself and aphidicolin

JU56 cells have been exposed to 1-β-d-arabinofuranosylcytosine (ara-C) in S phase, and again to aphidicolin (APC) or ara-C during G₂, and examined for chromosomal aberrations at c-metaphase. It was found that the two exposures acted synergistically in the production of chromosomal lesions of both the chromatid and isochromatid type. The results were interpreted as indicating that inhibition of the G₂ repair system prevented the repair of DNA single-strand regions produced by the incorporation of ara-C during semiconservative DNA synthesis.     

Calsyntenin-3 Molecular Architecture and Interaction with Neurexin 1α

Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca²⁺, and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca²⁺ and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function.     

Genomic instability associated with human LINE-1 rétrotransposition

LINE-1 (L1) retrotransposon accounts for approximately 17 % of the human genome. Because of the great number of identical copies, L1 can be implicated in genomic rearrangements associated with events of homologous recombination between heterologous sites. Moreover, even if the vast majority of the L1 elements are inactive, some are still able to mobilize themselves by retrotransposition. Thus, L1 is regarded as an insertional mutagenic agent. Moreover, recent works have shown that active retrotransposons were able to mobilize other sequences to generate retro-pseudogenes or to amplify other repeated sequences. Finally, L1 has been associated recently with new genomic rearrangements generated upon insertions such as large genomic deletions. L1 then can be considered as a major factor that has affected and shaped the human genome through several mechanisms.     

Immunomodulation with Recombinant Interferon-γ1b in Pulmonary Tuberculosis

Background

Current treatment regimens for pulmonary tuberculosis require at least 6 months of therapy. Immune adjuvant therapy with recombinant interferon-γ1b (rIFN-γb) may reduce pulmonary inflammation and reduce the period of infectivity by promoting earlier sputum clearance.

Methodology/Principal Findings

We performed a randomized, controlled clinical trial of directly observed therapy (DOTS) versus DOTS supplemented with nebulized or subcutaneously administered rIFN-γ1b over 4 months to 89 patients with cavitary pulmonary tuberculosis. Bronchoalveolar lavage (BAL) and blood were sampled at 0 and 4 months. There was a significant decline in levels of inflammatory cytokines IL-1β, IL-6, IL-8, and IL-10 in 24-hour BAL supernatants only in the nebulized rIFN-γ1b group from baseline to week 16. Both rIFN-γ1b groups showed significant 3-fold increases in CD4+ lymphocyte response to PPD at 4 weeks. There was a significant (p = 0.03) difference in the rate of clearance of Mtb from the sputum smear at 4 weeks for the nebulized rIFN-γ1b adjuvant group compared to DOTS or DOTS with subcutaneous rIFN-γ1b. In addition, there was significant reduction in the prevalence of fever, wheeze, and night sweats at 4 weeks among patients receiving rFN-γ1b versus DOTS alone.

Conclusion

Recombinant interferon-γ1b adjuvant therapy plus DOTS in cavitary pulmonary tuberculosis can reduce inflammatory cytokines at the site of disease, improve clearance of Mtb from the sputum, and improve constitutional symptoms.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00201123","term_id":"NCT00201123"}}NCT00201123     

Transfection of Corynebacterium glutamicum with Temperate Phage ϕ CG1

Versatile plasmid vectors useful for gene cloning in Brevibacterium lactofermentum, a glutamic acid-producing bacterium, have been constructed. The trimethoprim (Tp)-resistant dihydrofolate reductase gene derived from chromosomal DNA of the Tp-resistant mutant of B. lactofermentum was introduced into pAM330, a cryptic plasmid in B. lactofermentum. The constructed cloning vector pAJ228 (7.6kb) exists in 10 to 20 copies in cells of B. lactofermentum and donated Tp-resistance, which is a useful selective marker of transformants. pAJ228 was further improved to a versatile plasmid vector pAJ224 having some profitable characteristics such as smaller size (3.7 kb), higher copy number (60 ~ 80 copies), and additional useful cloning sites (Bam HI, Pst I and Sal I) equipped with two different promoters arranged at both orientations for the expression of passenger DNA without promoter. These plasmids were stably retained in B. lactofermentum even in the absence of Tp over many generations. Thus, they have been found very powerful vectors for gene cloning in Brevibacterium and the related bacteria.     

α-synuclein interacts with SOD1 and promotes its oligomerization

Background

Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are both neurodegenerative diseases leading to impaired execution of movement. α-Synuclein plays a central role in the pathogenesis of PD whereas Cu, Zn superoxide dismutase (SOD1) is a key player in a subset of familial ALS cases. Under pathological conditions both α-synuclein and SOD1 form oligomers and fibrils. In this study we investigated the possible molecular interaction of α-synuclein and SOD1 and its functional and pathological relevance.

Results

Using a protein-fragment complementation approach and co-IP, we found that α-synuclein and SOD1 physically interact in living cells, human erythrocytes and mouse brain tissue. Additionally, our data show that disease related mutations in α-synuclein (A30P, A53T) and SOD1 (G85R, G93A) modify the binding of α-synuclein to SOD1. Notably, α-synuclein accelerates SOD1 oligomerization independent of SOD1 activity.

Conclusion

This study provides evidence for a novel interaction of α-synuclein and SOD1 that might be relevant for neurodegenerative diseases.