L-Serine Deficiency and Asn133Ser Variant Quiz
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Questions and Answers

What was used to visualize the cytoskeleton in the cells?

  • Alexa Fluor 546
  • DRAQ5
  • Phalloidin CruzFluorTM488 (correct)
  • Vectashield antifade mounting media

    • How long were cells counterstained with DRAQ5 to visualize the nucleus?

  • 60 minutes
  • 10 minutes (correct)
  • 30 minutes
  • 20 minutes

    • Which software was used to prepare the images after acquiring confocal image stacks?

  • Fiji (ImageJ) (correct)
  • Adobe Photoshop
  • LEICA TCS software
  • GraphPad Prism

    • What was used as a negative control during imaging?

    <p>Samples without primary antibodies</p> Signup and view all the answers

    Which channel was used to analyze the overlapping fluorescence signals?

    <p>JACoP plugin of Fiji</p> Signup and view all the answers

    What role do astrocytes play concerning L-Ser?

    <p>They are responsible for the main production of L-Ser.</p> Signup and view all the answers

    What is the significance of the PP enzymes related to the nervous system?

    <p>They are linked to neurological phenotypes when genetically defective.</p> Signup and view all the answers

    Which substance does PSP preferentially dephosphorylate?

    <p>L-phosphoserine with higher affinity.</p> Signup and view all the answers

    Why is endogenous synthesis of L-Ser required?

    <p>There is limited permeability of L-Ser through the blood-brain barrier.</p> Signup and view all the answers

    Which superfamily does PSP belong to?

    <p>Haloacid dehalogenase-like superfamily.</p> Signup and view all the answers

    What is the primary function of L-Ser in neurons?

    <p>To act as an essential amino acid.</p> Signup and view all the answers

    In which location was native human PSP purified from?

    <p>Brain tissue.</p> Signup and view all the answers

    What characteristic of PSP supports its catalytic activity?

    <p>Its kinetic properties favor L-Ser production.</p> Signup and view all the answers

    What was the percentage decrease in Gly levels for the male patient in serum samples?

    <p>29.0%</p> Signup and view all the answers

    Which patient showed the highest decrease in D-Ser levels in serum samples?

    <p>F patient</p> Signup and view all the answers

    What was the change in the D/(D + L)-Ser ratio in fibroblast samples?

    <p>Significant decrease</p> Signup and view all the answers

    What is the Tm value for the wild-type PSP in the presence of 0.125 mM MgCl2?

    <p>58.5 °C</p> Signup and view all the answers

    What do Manders’ coefficients M1 and M2 measure?

    <p>The percentage of total signal from one channel that overlaps with another</p> Signup and view all the answers

    Which coefficient is used to determine the strength of the linear relationship between two channels?

    <p>Pearsons’ coefficient (r)</p> Signup and view all the answers

    During incubation, what was the Tm value for Asn133Ser PSP?

    <p>52.8 ± 0.1 °C</p> Signup and view all the answers

    How was ethics approval handled for the case study mentioned?

    <p>Ethics committee approval was waived due to the study's nature</p> Signup and view all the answers

    What is indicated by the k value for the wild-type PSP?

    <p>1.23 ± 0.04</p> Signup and view all the answers

    At what age did the proband first achieve independent walking?

    <p>19 months</p> Signup and view all the answers

    What happens to PSP activity during incubation at 37 °C with MgCl2?

    <p>Decreases then increases</p> Signup and view all the answers

    What condition was observed during the clinical examination of the proband at the age of three?

    <p>Spastic paraparesis with a dystonic component</p> Signup and view all the answers

    What developmental challenge did the proband face in school?

    <p>Difficulties that required special education</p> Signup and view all the answers

    How were the growth parameters of the proband at birth categorized?

    <p>Within normal range</p> Signup and view all the answers

    What measure helps in adjusting the threshold of channels in colocalization analysis?

    <p>Costes’ threshold</p> Signup and view all the answers

    What is the primary focus of the research conducted by Collet et al. in 1997?

    <p>The expression and sequence of human L-3-phosphoserine phosphatase</p> Signup and view all the answers

    How does calcium affect human phosphoserine phosphatase according to the research by Peeraer et al. in 2004?

    <p>It inhibits the magnesium-dependent enzyme</p> Signup and view all the answers

    Which disorder is directly related to serine biosynthesis as discussed by Jaeken et al. in 1996?

    <p>3-Phosphoglycerate dehydrogenase deficiency</p> Signup and view all the answers

    What is one finding reported by Ozeki et al. regarding phosphoserine phosphatase and schizophrenia?

    <p>Enzyme activity is elevated and correlates negatively with plasma D-serine concentration</p> Signup and view all the answers

    In the study conducted by Veiga-da-Cunha et al. on 3-phosphoserine phosphatase deficiency, what was primarily investigated?

    <p>Mutations causing the deficiency</p> Signup and view all the answers

    Which enzyme's structure was explored by Peeraer et al. in 2003?

    <p>Human phosphoserine phosphatase</p> Signup and view all the answers

    What was a significant aspect discussed in the review by Tabatabaie et al. regarding L-serine?

    <p>L-serine synthesis in the central nervous system</p> Signup and view all the answers

    Which biological activity was characterized by Veeranna & Shetty in 1990?

    <p>Phosphoserine phosphatase of human brain</p> Signup and view all the answers

    What was the melting temperature (Tm) of Asn133Ser PSP?

    <p>52.8 ± 0.1 °C</p> Signup and view all the answers

    What effect does the Asn133Ser substitution have on the stability of PSP?

    <p>It destabilizes PSP structure.</p> Signup and view all the answers

    During the incubation of Asn133Ser PSP at 37 °C, what was observed over time?

    <p>The secondary structure content decreased.</p> Signup and view all the answers

    How was the stability of wt PSP assessed in comparison to Asn133Ser PSP?

    <p>By comparing Tm values and CD spectroscopy.</p> Signup and view all the answers

    What was the significant observation regarding the thermal transition slope of Asn133Ser PSP?

    <p>It was significantly lower than that of wt PSP.</p> Signup and view all the answers

    Which condition was used during the incubation of the enzyme At 37 °C?

    <p>Potassium phosphate buffer pH 7.0 with 0.125 mM MgCl2.</p> Signup and view all the answers

    What change in CD spectra indicates an alteration in the secondary structure of a protein?

    <p>Decrease in ellipticity at 208 nm and 222 nm.</p> Signup and view all the answers

    What physiological role does Mg++ play concerning PSP?

    <p>It is a known ligand and activator.</p> Signup and view all the answers

    Study Notes

    L-Serine Deficiency and Asn133Ser Variant

    • L-serine is a non-essential amino acid crucial for numerous metabolic pathways, particularly in the brain, where its levels primarily originate from the phosphorylated pathway (PP).
    • The serinosome, a cytosolic metabolon, comprises enzymes responsible for L-serine synthesis from the glycolytic intermediate D-3-phosphoglycerate.
    • Phosphoserine phosphatase (PSP) catalyzes the final and irreversible step in the PP.
    • A homozygous missense variant (Asn133Ser) in the PSPH gene (encoding PSP) was identified in two siblings with a neurodevelopmental syndrome and myelopathy.
    • The Asn133Ser variant enzyme exhibits similar protein conformation, dimerization, and activity compared to the wild-type enzyme; however, it is less stable.

    Biochemical Properties of Asn133Ser PSP

    • The Asn133Ser variant's protein structure is not significantly altered, as indicated by far-UV circular dichroism (CD) spectra.
    • Both wild-type and Asn133Ser PSP exist as dimers (50 kDa homodimer protein), as determined via size exclusion chromatography (SEC).
    • Kinetic parameters (kcat, Km, kcat/Km) for the Asn133Ser variant differ slightly, demonstrating a decreased kcat but higher substrate affinity (lower Km), resulting in the same catalytic efficiency as wild-type.

    Patient Fibroblast Studies and Colocalization

    • Affected patient fibroblasts exhibited a significant reduction in the levels of PP enzymes (PHGDH, PSAT, PSP) compared to healthy controls.
    • The Asn133Ser variant showed increased perinuclear aggregates formation in patient cells.

    Serine and Glycine Levels in Serum and Fibroblasts

    • Significant decreases in L-serine, D-serine, and glycine levels were observed in both serum and fibroblast samples from patients compared to healthy controls.
    • These alterations suggest a disruption of the serinosome function and associated pathways.

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    L-Serine Deficiency PDF

    Description

    Explore the critical role of L-serine in metabolism and its implications in neurodevelopmental syndromes. This quiz delves into the biochemical properties of the Asn133Ser variant of the phosphoserine phosphatase gene and its impact on enzyme stability and function. Test your understanding of amino acids, enzyme mutations, and their physiological relevance.

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