A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI, the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca(2+).Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.We propose that W542 and Ca(2+) are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca(2+) is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis

Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [32P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs

De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome

Activating mutations in genes encoding phosphatidylinositol 3-kinase (PI3K)-AKT pathway components cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH, OMIM 603387). Here we report that individuals with MPPH lacking upstream PI3K-AKT pathway mutations carry de novo mutations in CCND2 (encoding cyclin D2) that are clustered around a residue that can be phosphorylated by glycogen synthase kinase 3β (GSK-3β). Mutant CCND2 was resistant to proteasomal degradation in vitro compared to wild-type CCND2. The PI3K-AKT pathway modulates GSK-3β activity, and cells from individuals with PIK3CA, PIK3R2 or AKT3 mutations showed similar CCND2 accumulation. CCND2 was expressed at higher levels in brains of mouse embryos expressing activated AKT3. In utero electroporation of mutant CCND2 into embryonic mouse brains produced more proliferating transfected progenitors and a smaller fraction of progenitors exiting the cell cycle compared to cells electroporated with wild-type CCND2. These observations suggest that cyclin D2 stabilization, caused by CCND2 mutation or PI3K-AKT activation, is a unifying mechanism in PI3K-AKT–related megalencephaly syndromes

Homo- and Heterosubtypic Low Pathogenic Avian Influenza Exposure on H5N1 Highly Pathogenic Avian Influenza Virus Infection in Wood Ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations

Emergence and spread of two SARS-CoV-2 variants of interest in Nigeria.

Identifying the dissemination patterns and impacts of a virus of economic or health importance during a pandemic is crucial, as it informs the public on policies for containment in order to reduce the spread of the virus. In this study, we integrated genomic and travel data to investigate the emergence and spread of the SARS-CoV-2 B.1.1.318 and B.1.525 (Eta) variants of interest in Nigeria and the wider Africa region. By integrating travel data and phylogeographic reconstructions, we find that these two variants that arose during the second wave in Nigeria emerged from within Africa, with the B.1.525 from Nigeria, and then spread to other parts of the world. Data from this study show how regional connectivity of Nigeria drove the spread of these variants of interest to surrounding countries and those connected by air-traffic. Our findings demonstrate the power of genomic analysis when combined with mobility and epidemiological data to identify the drivers of transmission, as bidirectional transmission within and between African nations are grossly underestimated as seen in our import risk index estimates

Homozygous intragenic deletion in the WT1 gene in a sporadic Wilms' tumour associated with high levels of expression of a truncated transcript

We have examined a panel of 21 sporadic Wilms' tumours for rearrangements in the Wilms' tumour suppressor gene, WT1. In one tumour with specific allele loss in chromosome 11pl3, a homozygous deletion in the 3′ end of the gene, encompassing exon 10 and the 3′ untranslated region, was identified. High levels of a truncated WT1 transcript, predicted to encode a polypeptide missing the fourth zinc finger were expressed in this tumour. All other samples showed normal patterns of digestion on Southern Hots. This observation confirms previous findings that large deletions in the gene occur infrequently in sporadic Wilms' tumours and that the zinc‐finger region of the encoded polypeptide is critical for correct functioning of the gene.© 1995 wiley‐Liss, Inc. Copyrigh

Release of MAP1A and MAP2 into the soluble fraction after phosphorylation.

<p>(<b><i>A</i></b>) <i>Release of MAP1A</i>. CCVs (20 µg) were phosphorylated and ultracentrifuged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g001" target="_blank">Fig. 1</a> except for using 2 mM cold ATP and with or without GST-Dyrk1A⁴⁹⁷. The resultant supernatants were blotted with anti-MAP1A antibody <i>(M1A).</i> (<i>n = 3</i>). (<b><i>B</i></b>) <i>Phosphorylation-dependent dissociation of MAP1A and MAP2.</i> CCVs were incubated as in <i>(A)</i> in duplicate tubes containing ATP and either wild type <i>(wt)</i> or double mutant (<i>DF</i>) GST-Dyrk1A⁴⁹⁷. After adding EDTA, one set of tubes was kept on ice, whereas the other set was ultracentrifuged to collect the soluble fractions. Aliquots of whole mixtures (<i>W</i>) and the supernatants (<i>S</i>) were subjected to immunoblotting. Both MAP1A <i>(M1A)</i> and MAP2 <i>(M2)</i> were identified in the same lane by blotting the PVDF membrane first with anti-MAP2 then with anti-MAP1A antibodies. (<i>n = 1, various preliminary performances carried out to lead the final assay conditions are not included</i>).</p

Effect of Dyrk1A on dissociation of adaptor proteins from CCVs.

<p><i>(</i><b><i>A</i></b><i>) Effect of phosphorylation.</i> CCVs (3.5 µg) were incubated with ±1 mM cold ATP and ± Dyrk1A (0.5 µg), as indicated in the panel, and ultracentrifuged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g001" target="_blank">Fig. 1</a>. The resultant supernatants were immunoblotted (<i>WB</i>) using the antibodies indicated in the figure. (<i>n = 2</i>). <i>(</i><b><i>B</i></b><i>) Release of α- and β-adaptins by Dyrk1A without phosphorylation</i>. CCVs (3.75 µg) were incubated with various amounts of GST-Dyrk1A⁴⁹⁷, but without ATP, either in kinase buffer (pH 7.4) or MES buffer (pH 6.5) containing 0.1 M NaCl, 5 mM MgCl₂. Appropriate amounts of GST were added into each tube to compensate for the different kinase amounts. After chilling on ice, the reaction mixtures were ultracentrifuged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g001" target="_blank">Fig. 1</a>. The supernatants were blotted by using anti-α- or β-adaptin antibody. (<i>n = 2</i>). <i>(</i><b><i>C</i></b><i>) Release of multiple CCV proteins by Dyrk1A without phosphorylation.</i> The same samples from (<i>B</i>) at pH 7.4 were analyzed by using various antibodies as indicated. <i>Lanes</i> (−) and <i>1–5</i> represent the kinase concentrations at 0, 0.1, 0.25, 0.5, 1.0, and 2 µg/assay. One µg Dyrk1A/assay is equal to 4.2×10⁻⁷ M by assuming the enzyme purity as 100%.</p

Proposed Dyrk1A functions in regulating synaptic vesicle endocytosis.

<p>Dyrk1A phosphorylated AP180 in cytosol. The phosphorylated AP180 may decrease its binding affinity to the AP2 complex; we hypothesize here that the decrease in such binding affinity would reduce recruitment of clathrin at the <i>nucleation</i> and <i>invagination</i> sites. Phosphorylation of dynamin 1, amphiphysin 1, and synaptojanin 1 at their PRD reduces the interaction between the PRDs and the SH3 domains of amphiphysin and endophilin. This likely slows down the <i>invagination</i> and <i>fission</i> steps of synaptic vesicle formation. Once endocytosed, the vesicle-associated proteins are quickly removed from the membranes (<i>uncoating</i>). The Dyrk1A-mediated phosphorylation releases first AP180 and β-adaptin from the vesicle membranes, while both α- and μ-adaptin subunits remain bound with the membranes. Additional factor(s) are required to release the membrane-bound subunits. Clathrin release from the vesicles is independent from Dyrk1A. We speculate that the adaptin subunits released in cytoplasm may reassemble into the AP2 complex after dephosphorylation.</p

Ratios of adaptin subunits recovered in cytosol to the precipitated fractions from cultured cells.

<p>Cytosol (<i>Sup</i>) and precipitated (<i>Ppt</i>) fractions from CHO and PC12 cells were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. SDS-PAGE was carried out by applying equal volumes of the soluble and insoluble fractions in each lane side-by-side. Immunoblotting was performed by using corresponding antibodies derived from mouse and rabbit. The first blots with mouse and rabbit antibodies were stripped (stripping buffer, PIERCE) and re-blotted with rabbit and mouse antibodies, respectively, for detecting other adaptin subunits in the same membranes. The antibodies used were mouse monoclonal antibodies against anti-α and γ-adaptins, rabbit monoclonal anti-β-adaptin antibody, and rabbit polyclonal anti-μ adaptin antibody. Each adaptin band in the <i>Sup</i> and <i>Ppt</i> was scanned, and the <i>Sup</i> to <i>Ppt</i> ratio was calculated. Four independent samples per each cell type were shown.</p