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 (A)</p> Signup and view all the answers

Which channel was used to analyze the overlapping fluorescence signals?

<p>JACoP plugin of Fiji (B)</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. (A)</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. (B)</p> Signup and view all the answers

Which substance does PSP preferentially dephosphorylate?

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

Which superfamily does PSP belong to?

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

In which location was native human PSP purified from?

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

What characteristic of PSP supports its catalytic activity?

<p>Its kinetic properties favor L-Ser production. (C)</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% (D)</p> Signup and view all the answers

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

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

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

<p>Significant decrease (B)</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 (D)</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 (B)</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) (B)</p> Signup and view all the answers

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

<p>52.8 ± 0.1 °C (B)</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 (D)</p> Signup and view all the answers

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

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

At what age did the proband first achieve independent walking?

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

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

<p>Decreases then increases (A)</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 (D)</p> Signup and view all the answers

What developmental challenge did the proband face in school?

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

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

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

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

<p>Costes’ threshold (B)</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 (C)</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 (A)</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 (D)</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 (B)</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 (C)</p> Signup and view all the answers

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

<p>Human phosphoserine phosphatase (B)</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 (C)</p> Signup and view all the answers

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

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

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

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

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

<p>It destabilizes PSP structure. (D)</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. (C)</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. (C)</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. (C)</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. (B)</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. (A)</p> Signup and view all the answers

What physiological role does Mg++ play concerning PSP?

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

Flashcards

Cell cytoskeleton visualization

Visualizing the cell's internal framework using Phalloidin CruzFluorTM488 conjugate.

Nuclear staining method

DRAQ5 fluorescent dye labels the cell nucleus for visualization.

Confocal microscopy

A microscopy technique that creates detailed 3D images of cell components.

Secondary antibodies role

These antibodies bind to the primary antibodies to enhance signal and visualization.

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Image preparation methods

Fiji (ImageJ) software is used for stacking and merging images acquired from different channels.

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L-Serine permeability

L-Serine has limited ability to pass through the blood-brain barrier.

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Endogenous synthesis

The body produces L-Serine itself, not just from outside sources.

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Astrocyte L-Serine production

Astrocytes are the primary cells responsible for creating L-Serine.

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Essential amino acid for neurons

L-Serine is important for the proper functioning of nerve cells.

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PP enzymes

Enzymes involved in producing L-Serine in the central nervous system.

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PSP enzyme's key role

PSP catalyzes the reaction that drives L-Serine production.

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Haloacid dehalogenase-like superfamily

A group of enzymes that PSP belongs to.

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Phosphoserine dephosphorylation

The removal of a phosphate group from phosphoserine to create L-Serine.

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Manders' Coefficients

Measures how much two signals overlap in an image. M1 shows how much of the signal in one channel overlaps with the other, while M2 does the same but with the channels reversed.

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Pearson's Coefficient

A statistical measure showing how strongly two signals are linearly related in an image. A high value suggests a strong linear relationship.

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Colocalization Parameters

These are measures used to quantify how much two different signals overlap in an image. This helps to understand if the signals are coming from the same location or different locations.

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Ethics Waiver

A decision by an ethics committee to exempt a study from full ethical review because it doesn't pose significant risks.

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Informed Consent

A process where individuals are given information about a study and agree voluntarily to participate.

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Clinical Evaluation

The process of assessing a person's health condition through observation, tests, and medical history.

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Spastic Paraparesis

A condition where muscles in the legs become stiff and difficult to control, leading to problems with walking.

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Dysarthria

Difficulty speaking clearly due to problems with muscle control involved in speech.

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PSP's Thermal Stability

The ability of PSP to withstand heat and maintain its structure. It measures how much temperature change is needed to unfold the protein.

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Asn133Ser Mutation Effect

Replacing the amino acid Asn at position 133 with Ser in PSP reduces the protein's stability and makes it unfold at a lower temperature.

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Tm of PSP

The melting temperature of PSP is the point at which it unfolds, transitioning from a stable state to a less stable one.

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Cooperative Unfolding

When a protein unfolds in a coordinated and synchronized manner, like a zipper closing, this is called cooperative unfolding.

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Mg++ as PSP Activator

Magnesium ions (Mg++) act as a helper or activator for PSP, making it function better.

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Physiological Mg++ Concentration

The normal level of magnesium ions (Mg++) found in human astrocytes, a type of brain cell.

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SDS-PAGE Analysis

A technique used to separate proteins based on their size. It helps identify different proteins and their abundance.

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Secondary Structure Alteration

Changes in the folding pattern of PSP, such as alpha-helices and beta-sheets, indicating potential loss of function.

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D-Serine Levels in F Patient

The F patient shows the most significant decrease in D-Serine levels, particularly in their serum samples.

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D/(D+L)-Ser Ratio in Fibroblasts

Fibroblasts from the F patient have a significant reduction in the ratio of D-Serine to total Serine (D+L), suggesting a decrease in D-Serine production.

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Glycine Levels in Serum

Both the M and F patients exhibit a marked reduction in Glycine levels in their blood serum.

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Glycine Levels in Cells

The M patient shows a noticeable 6% decrease in Glycine within their cells, indicating a change in intracellular Glycine levels.

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Asn133Ser PSP: Thermal Stability

The Asn133Ser variant of the PSP enzyme has a lower melting point (Tm) compared to the wild-type, suggesting weaker thermal stability.

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Asn133Ser PSP: Secondary Structure Stability

The Asn133Ser variant exhibits a faster rate of secondary structure degradation compared to the wild-type PSP, indicating decreased structural stability.

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Asn133Ser PSP: Activity

The Asn133Ser variant of PSP shows a decreased enzymatic activity over time, indicating a loss of function compared to the wild-type enzyme.

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D-Serine, L-Serine, and Glycine Levels

The study investigates the levels of D-Serine, L-Serine, and Glycine in serum and cellular samples, indicating changes in these amino acids in relation to a specific condition or treatment.

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PSP Function

PSP (Phosphoserine Phosphatase) is an enzyme that removes a phosphate group from phosphoserine to create L-serine, a crucial amino acid for brain function.

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L-Serine's Importance

L-serine is a crucial amino acid for brain function. It helps build proteins, supports nerve cell communication, and plays a role in neurotransmitter production.

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Astrocyte Role

Astrocytes, star-shaped brain cells, are the primary producers of L-serine in the brain.

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PSP Deficiency

A deficiency in PSP can lead to 3-phosphoserine phosphatase deficiency, a rare genetic disorder that affects L-serine production in the body.

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PSP's Role in Schizophrenia

PSP activity has been linked to schizophrenia, with some studies showing increased PSP activity in patients with the condition.

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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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Related Documents

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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