WEEK 2 (STUDY GROUP) Flashcards
How does glucose enter the cell?
Facilitated diffusion through membrane transporters for glucose (the GLUT family)
What are the substrates and products of glycolysis?
1 glucose –> 2 pyruvate, 2 ATP (4 ATP is made, but 2 ATP are used) , 4 e (as NADH)
What gets glucose to glucose-6-phosphate?
Hexokinase + ATP
Where does glycolysis occur?
Cytosol
What gets fructose 6 phosphate to F-1,6-BP?
Phosphofructokinase-1, (PFK-1), ATP
What does fructose 1,6 BP break into and how?
DHAP (3 carbon) and G3P (3 carbon) through aldolase
Between DHAP and GA3P, which way does equilibrium lean?
Towards DHAP
After DHAP and GA3P is produced, there is another phosphorylation and there is rearranging to PEP. How does PEP convert into Pyruvate?
Pyruvate Kinase (phosphorylation), ATP
What happens to pyruvate in anaerobic conditions?
Pyruvate is reduced to lactate, catalyzed by a dehydrogenase and NADH donates its electrons. It happens in the cytosol. This produces the NAD + we need to continue oxidizing glucose!
What happens to glycolysis in aerobic conditions? How is NAD+ made?
A shuttle moves the NADH/FAD2H to the mitochondria where it is reoxidized to NAD+, thus allowing, it to continue oxidizing glucose. Either a glycerol 3 phosphate shuttle or a malate aspartate shuttle is used.
How does hexokinase regulate glycolysis?
It is product inhibited by glucose 6-P. It will downregulate the pathway from glucose to glucose 6 P, when there is already enough G6P
How does PFK 1 regulated? (PFK 1 is the enzyme that phosphorylates F6P to F1,6-BP)
It is upregulated by AMP and F-2,6-BP. F-2,6,BP is high in a well fed state, when insulin is high. When the state is well fed, then glycolysis will be favored. PFK-1 is downregulated by ATP and citrate (when ATP is already high, more is not neeeded)
How is the step from PEP to Pyruvate regulated?
It is upregulated by F-1,6-P (a product from earlier in the glycolysis pathway that ensures glycolysis continues downstream). It is downregulated by increased amounts of ATP.
How is fructose oxidized? How does it enter glycolysis cycle to make ATP?
Fructose –> Fructose 1 P (through kinase) –> DHAP and GAP (through aldolase). Then GAP –> GA3P (kinase), now it can enter glycolysis at the GA3P stage or the DHAP stage.
How is galactose oxidized? How does it enter glycolysis? (First Step. Galactose –> _____?)
Galctose 1-P through GALK (kinase) and ATP
How is galactose oxidized? How does it enter glycolysis? (2nd Step. Galactose –> Galactose 1-P –> _____?)
G1P –> UDP Galactose through GALT (transferase)
How does UDP Galactose –> UDP Glucose?
GALE (epimerase)
How is galactose oxidized? How does it enter glycolysis? (Galactose –> Galactose 1-P –> UDP Galactose –> UDP Glucose –> _____?)
Glucose 1-P, then Glucose 6-P and enters glycolysis
What are some of the biosynthetic functions of glycolysis?
Can make 5 C Sugars, Amino acids, Fatty acids
What is glycation?
Non enzymatic addition of reducing sugars to proteins
What are the 2 functions of the pentose phosphate pathway?
Generate NADPH (by reducing NADP). And to protect against reactive oxygen
When and where does glucogenesis occur?
In the liver, during fasting/starvation/low carb diet. More glucose is needed
Glucogenesis is the opposite of glycolysis. Except for one step which can’t be easily reversed. Which step is that and how does it occur instead?
Pyruvate –> PEP! Pyruvate goes into Mitochondria instead where it is converted into OAA through Carboxylase, CO2 and ATP.
How does OAA taken out of cytoplasm?
Converted into malate through transporter; converted into Asp through transporter; Converted straight into PEP (through PEPCK)
What is glycogen?
Storage form of glucose
How is glucose synthesized? What enzymes are used?
Glycogen synthase is the addition of glucose through activated intermediate UDP-glucose, and transferase is used
How is glucose degraded? What enzymes are used?
Glycogen phosphorylase removes single glucose molecules, a debranching enzyme moves 3 piece glucoses to the end of the chain
variant
single specific difference between genetic sequence of 2 ppl
tandem duplication
copy next to it
interspersed duplication
copy elsewhere
copy number variant
multiple copies in a row, varies in length
silent/synonymous
AA (amino acid) does not change
nonsynomous
a single AA is changed
nonsense
early stop codon
splice variant
disrupts splicing motif, severe but variable effects, cuases various splice errors like exon skipping on intron retention
loss of function
partial or total reduction of protein levels
gain of function
allele causes protein to function in a new way instead of the normal way (eg. TF activates wrong gene, or a signalling protein is always on)
exome
the entire protein coding of the genome, aka all the exons, only 1% of the human genome
what is the classification for variants
benign, likely benign, uncertain significance, likely pathogenic, pathogenic
- Why are genotypes described using 2 letters (i.e. C/C or C/G)?
Genotypes are described always using 2 letters because humans have 2 copies of every chromosome, one maternal and one paternal (another word for this is ‘diploid’). The 2 genotype denotes whether a variant is homozygous (both alleles the same) or heterozygous (one of each allele).
- What kinds of variants are most likely to result in a Loss of Function?
Loss of Function means an allele that is not able to make any protein of any function. This is usually caused by deletions (whole gene or large segments are missing from genome), or variants that result in a premature stop codon (nonsense, frameshift, some splice variants). These variants are sometimes referred to as ‘truncating’ variants.
- How might a single missense variant cause disease?
A single missense variant might cause disease through a Gain of Function – for example, causing a protein to misfold, which distorts the structure of the cell, which causes disease (this is what happens in Sickle Cell Disease). A single missense variant could also cause disease through Loss of Function – for example, a variant in a critical protein region that prevents a protein from binding to any partner (this happens in some forms of Hereditary Breast/Ovarian Cancer
What types of work is ATP hydrolysis used to power, and what are examples of each?
Mechanical (ex: conformational changes in proteins), transport (ex: ion pumps), biochemical (ex: coupling favorable & unfavorable reactions; generating activated intermediates; protein modification)
What is an oxidation-reduction reaction?
Chemical reaction that involves a transfer of electrons between 2 species
What does positive vs negative delta G for a reaction mean?
negative = spontaneous reaction, positive = non-spontaneous
What does delta G depend on?
substrate and product concentrations
What is the major cell type affected in Gaucher disease?
macrophage
What are the 3 major organ systems affected in Gaucher disease?
liver, spleen, bone marrow
What is the treatment for Gaucher disease?
enzyme replacement therapy: administer glucocerebrosidase every 2 weeks
What is the pathophysiology of Gaucher disease?
deficiency of glucocerebrosidase (breaks down glucocerebroside into glucose and ceramide) –> results in accumulation of glucocerebroside in macrophages
What are the sources of acetyl-CoA?
fatty acids, ketone bodies, pyruvate, ethanol
What reaction does pyruvate dehydrogenase complex catalyze?
pyruvate + CoASH + NAD+ –> acetyl-CoA + CO2 + NADH + H+ (pyruvate is oxidized and decarboxylated)
What is the mechanism for regulation of pyruvate dehydrogenase complex?
phosphorylation by pyruvate dehydrogenase kinase (PDK) inactivates PDC; dephosphorylation by pyruvate dehydrogenase phosphatase (PDP) activates PDC
What molecules activate PDC?
ADP and pyruvate inhibit PDK –> activates PDC, Calcium (released during muscle contraction) activates phosphatase –> activates PDC
What molecules inhibit PDC?
Acetyl CoA and NADH inhibits PDC (product inhibition)
What is the energy yield for TCA cycle?
3 NADH = 7.5 ATP, 1 FADH2 = 1.5 ATP, 1 GTP
List the steps of the TCA cycle
Acetyl CoA + oxaloacetate –> citrate –> isocitrate –> alpha-ketoglutarate –> succinyl CoA –> succinate –> fumarate –> malate –> oxaloacetate
What steps of the TCA cycle reduce NAD+ to NADH + H+?
isocitrate –> alpha-ketoglutarate
alpha-ketoglutarate –> succinyl CoA
malate –> oxaloacetate
What steps of the TCA cycle reduce FAD to FADH2?
succinate –> fumarate
What steps of the TCA cycle generate GTP?
succinyl CoA –> succinate
What is the main regulatory enzyme of the TCA cycle, and how is it regulated?
isocitrate dehydrogenase (isocitrate –> alpha-ketoglutarate)
- Activated by: ADP, calcium
- Inhibited by: NADH
where does the TCA cycle occur?
Mitochondrial matrix
What enzyme catalyzes the reaction: oxaloacetate + acetyl CoA –> citrate? How is it regulated?
Citrate synthase
- activated by low ATP levels
- inhibited by citrate
What enzyme catalyzes the reaction: alpha-ketoglutarate –> succinyl CoA? How is it regulated?
alpha-ketoglutarate dehydrogenase
- activated by calcium
- inhibited by NADH
What enzyme catalyzes the reaction: malate –> oxaloacetate? How is it regulated?
Malate dehydrogenase
- inhibited by NADH
what are the biosynthetic functions of the TCA cycle?
- Oxaloacetate –> amino acid synthesis
- Malate –> gluconeogenesis
- Citrate –> FA synthesis
- Alpha-ketoglutarate –> amino acid synthesis, neurotransmitter (brain)
- Succinyl-CoA –> heme synthesis
What is glutaminolysis?
Glutaminolysis is a process active in most proliferating cells, and especially tumor cells, that uses glutamine (most abundant amino acid in the plasma) to enter the TCA cycle
- Glutamine is transported into the mitochondria –> converted to glutamate –> converted to alpha-ketoglutarate –> enters the TCA cycle
Describe the structure of the mitochondrial membranes. Why are these important for ATP generation?
- Outer membrane- highly permeable due to VDACs (voltage gated ion channels, sensitive to membrane potential)
- Inner membrane- highly impermeable to nucleotides and ions. Only voltage-sensitive translocases use energy from electrochemical gradient for transport.
**this inner membrane impermeability is what uniquely allows ATP synthase to harness the electrochemical pH gradient due to lack of H+ in matrix to catalyze formation of ATP.
Where does Complex 2 of the ETC receive its electrons from?
- e- from Glycerol-3-phosphate shuttle
- e- from TCA cycle (succinate dehydrogenase)
- e- from Beta-oxidation of FA (ETF: Q oxidoreductase)
*Note: remainder of complexes receive e- from glycolysis
What is the purpose of the glycerol-3-phosphate shuttle in the ETC?
Main mechanism of transporting electrons into the mitochondrial matrix for ETC
DHAP + NADH (outside)–>Glycerol-3-Phosphate–>DHAP (outside) +FADH2 (inside)
What is the key regulator of Oxygen consumption in the ETC?
[ADP] (Cannot make more ATP without ADP)
Counter regulation point: If ETC slows down, TCA cycle will also slow down due to slower production of NAD in ETC
What are two medically relevant compounds which use “uncoupling” of the ETC to harness their effects? How do they do this?
- Thermogenin- allows H gradient to dissipate while ETC runs; use this energy to create HEAT instead of ATP (think:babies and brown fat)
- 2,4-dinitrophenol (2,4-DNP)- binds H outside mitochondria and carries them back inside the matrix. Keeps the ETC going, sold as dangerous fat burning drug
How does the malate-aspartate shuttle work, and what is its purpose?
Less common mechanism to transport electrons into ETC
To enter: OAA + NADH –>malate (diffuses in) –> OAA + NADH
To exit: OAA +NH3 from glutamate (transanimase)–> Aspartate (diffuses out)
Where is Ubiquinone located in the membrane? What is its function in the ETC?
It is an Integral membrane protein within CoQ (only component NOT bound to protein)
CoQ is a mobile electron carrier that accepts e- from Complexes 1 & 2 and donates them to Complex 3.
How do the ETC complexes allow the transfer of electrons?
Redox potential of complexes increases along the ETC.
- NADH is a HIGH energy compund since it is the MOST reduced (low redox potential)
- Terminal electron acceptor, O2, is a LOW energy compound since it is the most OXIDIZED (high redox potential)
Where is Cytochrome C located in the membrane? What is its function in the ETC?
It is located in the intermembrane space.
Cyctochrome C has a heme-iron attachment to bind and release electrons from complex 3 to 4.
What is the mitochondrial permeability transition pore’s role in survival?
Pathological conditions like stroke/TBI-
Triggered by increased Calcium, Phosphate, or ROS; leads to opening of pore, swelling and apoptosis.
Lack of O2 (ischemia) can prevent ETC from functioning, so ATP is hydrolyzed to reverse the H gradient. With decreased ATP to prevent pore opening, cascade leading to apoptosis ensues.
What are common sources of ROS in the cell?
- CoQ
- Oxidases, peroxidases, oxygenases (cytochrome P450 enzymes)
- Ionizing radiation
What are some examples of enzymatic and nonenzymatic antioxidants cells use to protect against ROS damage?
- Enzymes- catalase (H2O2 in peroxisomes), superoxide dismutase (mitochondria) , glutathione peroxidase/redctase (harness NADPH from PPP)
- Non-enzymes- Vitamins C & E, carotenoids, melatonin etc.
What are the different mitochondrial membrane transport systems?
- Adenine nucleotide translocase- exchange ATP/ADP
- Pyruvate and Phosphate Symporters
- Calcium (Driven by electrochemical gradient inside)
- Others- Phosphate/malate, citrate/malate, aspartate/glutamate, malate/alpha-ketoglutarate
cytogenesis
The study of chromosomes and cell division
Regarding DNA structure, the double-stranded helix is wrapped around histones, creating basic structural units of DNA packaging called?
Nucleosomes
Histones are wrapped into a telephone wire-like shape called a?
Solenoid
Wrap solenoids are further wrapped into?
chromatin loops
Chromatin loops are held together by a ___ scaffolding to form the chromosome structure?
protein
What stage of the cell cycle to we often study chromosomes?
Metaphase of Mitosis
What reagent is a spindle fiber inhibitor? What stage in the cell cycle with this reagent inhibit?
Colcemid; Anapahse during which spindle fibers pull sister chromatids to opposite poles. Due the this reagent the cells will be arrested in metaphase, which precedes anaphase.
What are telomeres? What happens if you lose the telomeres?
End of chromosome made up of repeat sequences; chromosome will start to degrade and the cell will apoptose
The short arm of a chromosome is also called?
p arm, recall tool -“petite”
The longer arm of a chromosome is also called?
q arm
Metacentric chromosome
centrosome placed in the middle of the chromosome; p and q arms of relatively same sizes
Submetacentric chromosome
If centromere is more towards one end than the other
Acrocentric chromosome
centromeres on the end of the chromosomes; DNA that sticks off the end referred to as stalks and satellites- can delete with no clinical consequence
Formal definition: Acrocentrics (13, 14, 15, 21 and 22) are a special class of human chromosomes. They have very small p arm comprised of large, tandem arrays of rDNA genes (stalks; stain positive only by silver staining).
They associate in interphase to form the nucleolus (also called nucleolus organizer regions or NORs)
At end of stalks are “satellites” (which are made up of highly repetitive “junk” DNA and no known coding sequences)
Length/size of stalks and satellites are polymorphic
The banding pattern of a chromosome across different people [apart from resultant abnormalities] is (different/the same)?
the same because banding looks at global packaging of DNA in the chromosome and not the specific sequences
euploid
numerical chromosomal abnormality; # of chromosomes is an exact haploid set (23 chromosomes); addition or subtraction of haploid set
aneuploid
numerical chromosomal abnormality; loss or gain of whole chromosomes (not haploid set)
structural chromosomal formation: ring
telomere of the chromosome has been deleted and in order to retain the chromosomes integrity, it circularizes and re-ligates
Translocation
break on two different chromosomes and swapping of material; no net gain or loss of DNA
cytogenic nomenclature
number of chromosomes present, sex chromosome composition, descriptive characters of abnormalities (lowest number first). Example: 47, XX, +13 means this cell has a whole extra chromosome 13 in a female
Lyon hypothesis
In females one X chromosome is “active” (genes are transcribed and translated). One is “inactive” (remains condensed; Barr body in interphase)
X inactivation occurs when and how?
early in embryonic life (~2 weeks after fertilization); randomly, inactivating either the maternal or paternal X chromosome; X-activation is clonal (after one X chromosome have become inactivated in a cell, all of that cell’s descendants have the same inactive X); Mediated by the XIST gene; DNA of inactive chromosome also highly methylated
Meiosis
specialized form of cell division that occurs only during gametogenesis;
comprised of MI and MII
shuffles the genetic material through recombination
divides genetic material in half
Meiosis I
reduction division(46->23, 2n->1n, diploid->haploid) occurs only in meiosis
Meiosis II
identical to mitosis in somatic cells except only 23 chromosomes are present
Meiosis I: Prophase I - lepotene
chromosomes begin to condense
Meiosis I: Prophase I - zygotene
homologs align (synapse) and held together by synaptonemal complexes
Meiosis I: Prophase I - pachytene
each pair of homologs (bivalent) coils tightly, crossing-over occurs
Meiosis I: Prophase I - diplotene
homologs begin to separate, but remain attached at points of crossing-over (chiasmata)
Meiosis I: Prophase I - diakinesis
separation of homolog pairs, chromosomes are maximally condensed
Recombination
Number of chiasmata per chromosome correlates with chromosome size
Thought that at least one chiasma per chromosome arm is required for normal segregation
Only one sister chromatid is involved in each cross-over event
Female recombination > male recombination
Recombination decreases near centromeres in males & females
Recombination increases near telomeres in males & females
Pseudoautosomal Regions
X and Y chromosomes share TWO regions of homology which undergo very high levels of genetic recombination
Consequences of Meiosis
Random segregation during meiosis I makes the likelihood of any two gametes from an individual having the exact same chromosomes equal to 1 in 223 (1 in 8 million)
Shuffling of the DNA through recombination makes the above likelihood even smaller
That’s why no two people are exactly the same!!
Female vs. Male Meiosis: Commences
Male (puberty); Female (early embryonic life)
Female vs. Male Meiosis: Duration
Male (60-65 days); Female (10-50 years)
Female vs. Male Meiosis: # mitoses in gamete formation
Male (30-500); Female (20-30)
Female vs. Male Meiosis: Gamete produced per meiosis
Male (4 spermatids); Female (1 ovum and 2-3 polar bodies)
Female vs. Male Meiosis: Gamete production
Male (100-200 million per ejaculate); Female (1 ovum per menstrual cycle)
Reasons for constitutional chromosome analysis (prenatal)
Advanced Maternal Age
Family history of Down syndrome or other chromosome abnormality
Ultrasound anomalies
Abnormal screening tests
Reasons for constitutional chromosome analysis (postnatal)
Congenital heart defect
Multiple congenital anomalies
Mental retardation, mild to profound, of unknown origin or associated with minor or major malformations
Ambiguous genitalia
Primary amenorrhea
Multiple unexplained spontaneous miscarriages
Mechanisms of Aneuploidy (presence of an abnormal number of chromosomes in a cell)
Nondisjunction:
Failure of homologous chromosomes (MI) or sister chromatids (MII) to separate;
Trisomy and monosomy can originate from meiotic or mitotic nondisjunction;
Parental origin of the extra chromosome in trisomy is most often maternal and most often a result of a MI error.
Such errors increase in frequency with maternal age.
Errors of Meiotic Segregation
Non disjunction: trisomy/monosomy
Malsegregation of “abnormal” chromosomes:
translocations
Robertsonian translocation
Inversions
Robertsonian Translocation
Robertsonian Translocation - a translocation between two acrocentric chromosomes (13,14,15,21,22) which results in the loss of the short arms of both chromosomes, but does not affect the DNA content of the long arms.
Microdeletion Syndromes
Phenotypically and genetically characterized syndromes involve complex but recognizable phenotypes
Deletion of small region or band containing 10-100 genes (usually less than 5Mb)
Occur at a low incidence in the population
Occur sporadically although can be inherited in a dominant fashion
Clinical Uses of FISH Analysis
Detect numerical abnormalities using non-dividing cells (interphase) - using fluorescent probes
Detect abnormalities which are beyond the resolution of standard G-band analysis
Identify the chromosomal origin of unknown material ie “mar” or “add” material
Prophase
chromosomes condense and coil
nuclear membrane disappears
spindle fibers begin to form from centrioles
Metaphase
chromosomes reach their most condensed form
chromosomes align along the middle of the cell (equatorial plane)
spindle fibers begin to contract
Anaphase
centromere split
spindle fibers pull chromatids toward opposite sides of the cell (centromere first)
Telophase
two nuclear membranes form spindle fibers disappear
chromosomes decondense
