Nucleotide topics
8. Viruses, retroviruses and reverse transcription
Virus
a virus is an infectious, parasitic agent that can only replicate within other host cells
viruses does not really fit our definition of living organisms, as they are unable to replicate without a host cell
all viruses have a genome and capsid. the viral genome can be single- or double stranded, DNA or RNA, linear or circular.
the capsid is a protein coat surrounding the genome, it consist of one protein, coded by only one gene
Retroviruses
some RNA viruses contains an RNA-dependent DNA polymerase called reverse transcriptase
On infection, the single stranded RNA genome and the enzyme enters the host cell
the reverse transcriptase first synthetize an DNA strand, complementary to the RNA strand, the primer for this reactions is tRNA lys
the RNA is then degraded except for a small portion, which acts as the primer for the synthesis of the second DNA strand
reverse transcriptase has 3 functions: RNA-dependent DNA pol., RNA degradation and DNA dependent DNA pol.
this resulting DNA duplex often becomes incorporated into the genome of the host cell, the integrated viral genes can be activated and transcribed, and the products, viral genome and proteins, can be packed as new viruses
the enzyme responsible for integration is integrase
LTR-Ψ-GAG-Pol-Env-LTR
most retroviruses contains the Ψ-GAG-Pol-env sequences
the LTR sequences are needed for initiation and regulation of transcription
the Ψ sequence is important for packing of retroviral RNA into mature viral particles
the sequence which contains the Gag (group associated antigen) and pol is translated into a large polyprotein which is cleaved into six proteins with distinct features
the gag gene proteins are structural proteins, the pol gene codes for integrase, protease and reverse transciptase
the env gene codes for the viral envelope proteins
Some retroviruses cause cancer and aids
some retroviruses contains an oncogene, that when expressed can cause the cell to grow abnormally → rous sarcoma virus is such a RNA-tumor virus
in addition to the typical retroviral genes, rous sarcoma virus also contains “src” which codes for a tyrosine-specific protein kinase, which is known to affect cell-division, cell-cell interactions
HIV causes AIDS, the tropism of HIV is the CD4 of T-cell
HIV genome contains many genes, other than the usual ones, like nef (ADP-ribosylation factor) involved in penetration, tat is a protein which “steals” transcription factors from the host, in order to express viral genes
drugs for aids:
AzT (azido dideoxy thymidine) reverse transcriptase, cause problems in bone marrow → anemia
dideoxyinosine (DDI) → protease inhibitor
Transposons
transposons or transposable element is a genetic sequence which can change position within the genome
45% of the genome is comprised of transposons.
there are 2 types: retrotransposons and DNA transposons
retrotransposons: “copy and paste”, these transposons are first transcribed to RNA, which is then reverse transcribed back into the DNA, which will incorporate into the genome
the retrotransposons themselves code for reverse transcriptase, which will reverse transcribe the RNA back into the DNA
DNA transposons are “cut and paste”, they simply relocate from one location in the genome to another
when transposons move around they can move into an exon and disrupt it, causing mutations, for this reason transposon are generally negative
eukaryotic cells use the RNA interference pathway to inhibit the activity of transposons
some aspect of transposons can also be beneficial
Telomeres
telomeres are located at the ends of a chromosome, they contain long (several thousands base pairs) non-coding repetitive sequences, in humans the repeating sequence is TTAGGG
with each cycle of DNA replication the chromosome is slightly shortened, the telomeres acts as expendable regions of the chromosome, preventing important genetic information from being lost after replication
in babies the telomeres are approx. 11.000 bp, in elderly this length is reduced to 4.000 bp. due to this it’s hypothesized that telomere length is related to ageing
telomerase is an enzyme which elongates the telomers, it is a special form of reverse transcriptase, and uses an internal RNA template to synthesize more telomeres
Homing
certains introns contains a gene for enzymes which promote homing (type I introns) or retrohoming (type II introns)
in type I an endonuclease is expressed, if allele a of gene x contains a homing intron while allele b doesn’t → endonuclease cuts the allele b at the site where the intron is in allele a → double strand break repair will insert a intron at the site
in type II a endonuclease/reverse transcriptase is needed → allele a of gene Y contains a type II intron while allele b doesn’t → the spliced intron (after transcription of aY) inserts itself and cuts the non-coding strand → it produces a self-complimentary DNA strand → the RNA is removed and the second strand is synthesized
9. Molecular tools of the regulation of gene expression with special respect to certain physiological and pathological conditions
Principles of gene regulation
only a fraction of the genes of an organism are expressed at any given time, the amount of gene product transcribed also depends on the gene
some proteins are present in very large amounts, like elongation factors used for protein synthesis, while others, like enzymes which repair rare DNA lesions, may only be present as a few molecules
some gene products are useless alone, and only useful when synthesized together with the products of other genes
imagine a cell which increases its transcription glucose-6-phosphatase, but not the transcription of other enzymes of gluconeogenesis
Different gene categories
the genome of an organism contains many genes, some genes are always expressed, while some are only expressed at certain times
according to this, we can distinguish between different gene “categories”
housekeeping genes, also called constitutive genes, are genes which are always expressed, these includes genes coding for proteins involved in transcription and translation, and central metabolic pathways
cell type-specific genes are genes which are only turned on in certain cell-types, which give each cell type its special properties and functions
e.g. only β-cells of the langerhans islets express the gene for insulin
developmental regulatory genes are genes which are turned on during certain stages of growth and development of an organism
e.g. the protein SRY, which is involved in determining the sex of the organism, is only expressed during a few days of fetal life
inducible genes, are genes which are normally turned off, but which are turned on in response to external stimuli
this includes genes which are activated in response to hormones, or genes involved in DNA repair
repressible genes are genes which are normally on, but which are turned off in response to external stimuli
e.g when bacteria have good supply of tryptophan, they repress the genes involved in tryptophan biosynthesis
Promoters
RNA polymerase initiate transcription when binding to promoters in the DNA
promoters are small segments of DNA upstream to the gene, the structure of promoters influence RNA polymerase’s affinity to the gene in question, thereby regulating the rate of transcription
promoters are usually AT-rich, common promotors include the TATA box and the CAAT box
regulatory proteins can enhance or interfere with the interactions between RNA-pol. and the promoter, thereby also influencing transcription
repressors interfere with these interactions, while activators enhance them
repressors bind to binding sites on the DNA called operators, which are generally between the promoter and the gene
when repressors are bound to the operator, RNA polymerase can’t move along the DNA, which is how they prevent transcription
activators binds to regions called enhancers, unlike operators, enhancers are often far away from the promoter
when activators bind to the enhancer, they increase the binding of RNA pol. to the promoter
receptors and activators aren’t always bound to their operators and enhancers - they’re only bound in response to a cellular signal.
this cellular signal, often a small molecule, changes the conformation of the regulatory protein, which causes it to bind to or dissociate from the DNA
RNA polymerase can switch out its σ-subunit depending on what needs to be expressed, changing the affinity of RNA polymerase to different promoters
The σ70 subunit binds to the promoter for housekeeping genes, and so σ70 subunit is the subunit which is active most of the time
during stress, the σ70 subunit is switched out with σ32 subunit, which changes the affinity of RNA pol. so that it binds to promotor of heat shock genes
DNA-protein binding
for protein like activators and repressors to bind to DNA they must have certain DNA-binding domains or motifs
the most important types of DNA-binding domains are helix-turn-helix, zinc fingers, homeodomain, leucine zipper, and helix-loop-helix
DNA-binding proteins must not only bind to DNA, but they must bind to very specific regions of DNA, i.e. a specific operator
for this to be possible, the DNA-binding proteins must have amnioacid side chains which bind to specific nucleotide sequences
these aa are often basic, which allows them to bind to the negatively charged DNA
for ex., glutamine and aspargine binds to adenine bases, arginine side chains bind to guanine bases
helix-turn-helix: 20 aa long domain, consisting of some ⍺-helix segments and a β-turn. it mostly found in bacteria, most notably in the Lac repressor
zinc finger: 30 aa long domain containing zinc ions, mostly found in eukaryotes, multiple zinc fingers are found in one DNA-binding protein
homeodomain: 60 aa long domain, mostly found in eukaryotes, but its similar to helix-turn-helix
leucine zipper: 60-80 aa long domain, the name comes from how every 7th aa is leucine, and that the residues intertwine like a zipper. found in the CREB transcription factor
helix-loop-helix is comprised of to amphipatic ⍺-helixes linked by a loop. this 50 aa domain is found in many transcription factors, most importantly HIF-1⍺
Gene regulation in bacteria
The lac operon
an operon is a cluster of genes that are controlled by the same promoters, and which are transcribed together
the best-known example of operons is the lac operon in E.coli. → this operon codes for genes needed for lactose metabolism, however, to save resources it should not be expressed when there is no lactose available for the bacteria
the lac repressor represses these genes when lactose is not available, when lactose is available the Lac repressor stops repressing these genes: so genes needed for lactose metabolism are exposed.
however, when both glucose and lactose are available lactose metabolism should be inhibited anyway, because glucose metabolism is more efficient than lactose metabolism
The SOS response
the SOS response in bacteria is a cellular response to great DNA damage, during the response the cell cycle is arrested and DNA repair is induced
E.coli. contains genes for many proteins that try to fix this DNA damage
however, during normal conditions, these genes are inhibited by a repressor called LexA
when DNA damage occurs, a protein called RecA binds to the damaged DNA
RecA then breaks down LexA, so that it doesn’t repress the SOS response genes, when this repression is broken, the genes are expressed, so the proteins can try to fix the DNA damage
Regulation of bacterial mRNA function
protein synthesis is not only regulated at the transcriptional level, but also at the translational level
bacterial mRNA can be regulated in two ways, trans and cis
every mRNA contains a site where ribosome binds to it
if this site is difficult to reach, then the mRNA has more trouble binding to the ribosome, so its translated less often, is the site is easy to reach, its translated more often
in trans regulation refers to when small RNA molecules bind to mRNA and either block or expose the ribosome-binding site
in Cis regulation involves mRNA which contain ligand-binding structures called aptamers, these aptamers can bind certain ligands, like TPP, glycine or AdoMet
when the aptamers bind these ligands, the mRNA coils, which makes the ribosome-binding site hard to reach, thereby inhibiting translation
Gene expression in eukaryotes
Differences in regulation of gene expression
Gene expression in eukaryotes is different then in prokaryotes on several levels
in bacteria, transcription and translation happens at the same time and place, while in eukaryotes they’re separated in both time and place
this means that they don’t take place at the same time, and not in the same cellular component
in bacteria, most genes are active while only certain ones are repressed. in eukaryotes, most genes are inactive, and only the ones needed are active
bacteria contains no histones, and have a different chromatin structure than eukaryotes
Epigenetics
refers to reversible heritable chemical modifications of DNA and histones, which regulate gene expression in eukaryotes
there are multiple mechanisms of epigenetics, as the most important being DNA-methylation, histone modification and nucleosome remodeling
epigenetics are influences by environmental factors, like diet, exercise, chemical, drugs..
they are a testament to how your parents lifestyle can affect you: not by changing the genes themselves but by altering their transcription pattern
DNA-methylation
DNA-methylation refers to the methylation of cytosine bases in promoter regions in DNA, converting them to 5-methylcytosine
this process typically decreases the affinity of RNA polymerase towards the promoter
thereby inhibiting gene transcription, this only occurs on cytosine bases in promoters called CpG islands
when the DNA strand is duplicated the new strand is methylated like the original
Histone modification
Recall that a nucleosome is the complex of DNA around a histone, these histones are involved in regulating the structure of chromatin
by covalently modifying the “tails” of the histones, the structure of chromatin can be altered
modification which cause DNA to wrap around the histone tighter decrease gene transcription, and vice versa
we say that the DNA coiling is relaxed or tightened
the histone codes refers to the patterns of covalent modification of the histones, of which there are billions of possible combinations
acetylation of a histone decreases the number of positive charges on the histone, thereby relaxing the DNA coiling, increasing transcription. deacetylation does the opposite
these processes are catalyzed by histone acetyltransferase (HAT) and histone deactylase (HDAC)
methylation of histones tightens the DNA coiling, decreasing transcription, this methylation occurs on the lysine residues of histones, of which there are many, this process is catalyzed by DNA methyltransferase (DNMT)
Nucleosome remodeling
certain proteins can remodel the nucleosomes, thereby changing the coiling of DNA to influnce gene expression
in eukaryotes the most important chromatin remodellers is the SWI/SNF protein complex
Hypoxia
In hypoxia, there is a lack of oxygen and the HIF-1⍺ cannot be constantly hydroxylated (req. vit.C) and it cannot form complexes with vHPL protein
the HIF-1⍺ acts as a TF for proteins for the hypoxia response, these proteins include: erythropoeitin, VEGF, GLUT and glycolytic enzymes among others
Oxidative stress
oxidative stress (ROS) can activate the NRF2 pathway. NRF2 is a TF which regulates expression of many genes related to protection of oxidative stress
in normal conditions NRF2 is bound to Keap1, which inhibits NRF2. Keap1 targets NRF2 for ubiquitination and degredation
when there is oxidative stress, Keap1 will inhibit NRF2 and it translocates to the nucleus
in the nucleus it forms heterodimers with Maf proteins and binds ti the ARE (antioxidant response element)
the mechanism of release from Keap1 is by oxidation of cys residues
upon binding of ARE it induces expression of oxidative stress protection, like genes for NADPH synthesis, glutathione synthesis, or other genes for elimination of ROS
a non-canonical way of activation has been discovered, where protein interaction with Keap 1 can release NRF2, these are p21, p62 and BRLA1
ROS can also activate NF-kB pathway. ROS activates IKKβ, which activates NF-kB. NF-kB will induce the transcription of many pro-inflammatory cytokines, like TNF⍺, IL-1, IL-6, COX2 and iNOS
ROS are also able to activate ASK1 kinase, this is a MAPK pathway leading to apoptosis
Inflammation
several cell-surface receptors will eventually lead to activation of NF-kB.
NF-kB can also be activated canonical (regular way) or non-canonically
NF-kB consist of two subunits (p50 and Rel A) which is inhibited by Ik-β⍺
from ligand binding to its receptor, IKKβ is activated which phosphorylates IKβ⍺
the active NF-kB can move to the nucleus and promote inflammatory protein
10. Biomolecules and molecular processes participating in the regulation of the cell cycle
Cell cycle
the cell cycle has 4 well-defined stages
S-phase: DNA is synthetized to create copies for both daughter cells
G2-phase: protein synthesize and cell growth to approx 2x size
M-phase: cell division
G1-phase: RNA synthesis and DNA synthesis / Go : cells stop dividing
Regulating the cell cycle
the cell cycle is regulated by oscillating values of specific protein kinases
these kinases phosphorylates protein involved in metabolic activities of the cell to control them, this ensures that the cell division proceeds correctly
these kinases are heterodimers that consist of two subunits: one cyclin subunit which regulates the activity, and one CDK subunit that has kinase activity
CDK doesn’t work without binding a cyclin
there are at least 10 different cyclin molecules and 8 CDK molecules
they combine in many combinations at specific points in the cell cycle
the cell produces specific combinations of cyclin and CDK during specific places to control the processes that should take place in each phase
During M-phase, Cyclin B and CDK 1 is produced by the cell, while other cyclins and CDK are inhibited or degraded, the CDK 1 - Cyclin B heterodimer then phosphorylates and activates proteins needed for mitosis
for this process to work, the correct cyclin and CDKs must be present, and the other cyclins and CDKs must be removed or inhibited
the cell uses 4 different mechanisms to regulate the cyclin and CDKs
phosphorylation/dephosphorylation of CDK
degradation of cyclin
periodic synthesis of CDK and cyclins
action of specific CDK-inhibiting proteins
Regulation of CDKs by phosphorylation
the activity of CDK can be tightly regulated by phosphorylation and dephosphorylation at two specific residues on the protein
phosphorylation at the 15th residue, a tyrosine (tyr15) deactivates CDK2, while phosphorylation at Thr160 by CAK (CDK-activating-kinase) activates CDK 2
dephosphorylation of Tyr15 by PTPase also activates CDK2
in case of single-strand breaks id DNA in a cell leads to the cell to stop in G2-phase
a specific protein is activated and leads to inactivation of PTPase, which means CDK2 stays inactive
when the repair is done, PTPase is activated and the cell cycle continues
Controlled degradation of cyclin
the process of mitosis requires first the activation and then destruction of cyclin A and B, which activates the M-phase CDK
these two cyclins contain an amino acid sequence called the “destruction box”
a protein called DBRP (destruction box recognizing protein) recognizes these destruction boxes and activates ubiquitin ligase, causing ubiquitination and proteolysis
during mitosis, cyclin A and B and M-phase CDK are activated
the CDK then phosphorylates DBRP which triggers destruction of Cyclin A/B which are activators of M-phase CDK → feedback regulation
Regulated synthesis of CDKs and cyclins
By regulating the synthesis of CDKs and cyclins, we can regulate their levels in the cell
Cyclin D, cyclin E, CDK 2 and CDK4 are only synthesized in the presence of the TF E2F
this TF is activated when growth factors and certain cytokines binds to the cell
the GF phosphorylates two proteins called Jun and Fos, which acts as TFs for E2F, the CDKs activated by it induce the synthesis of specific nuclear TFs for producing the enzymes needed for DNA synthesis, this allows the cell to enter the S-phase
Inhibition of CDKs
CDKs are inhibited by many different proteins, like p16, p21, p27, p53 and p57
these proteins, and other CDK inhibiting proteins, are also tumor suppressors: they prevent the cell from duplicating if something is wrong and by doing this, prevent tumor formation
INK4 protein family: p16(INK4a), p15(INK4B), p18(INK4C), p19(INK4D)
CIP and KIP protein family: p21(CIP1), p27(KIP1), p57(KIP2K)
What do CDK phosphorylate?
CDKs phosphorylate protein needed for cell division
the structure of the nuclear envelope is maintained by lamin
lamin is phosphorylated by CDK, ehich causes it to disintegrate which causes the nuclear envelope to break
after division, actin and myosin are phosphorylated and inactivated by CDK
another very important for CDK is retinoblastoma protein or pRb
when pRb is not phosphorylated it binds to the E2F and inhibits it, the E2F is then unable to promote transcription of proteins necessary for DNA replication, which makes the cell unable to pass from G1 → S-phase
The cyclin E / CDK 2 heterodimer can phosphorylate pRb which occur when the cell receives a signal to proceed with the cell division
when there is a ds-DNA break in DNA, the cell should not divide
a protein called MRN, binds to the ds-DNA break and activate two kinases called ATM and ATR → these will phosphorylate and activate p53
p53 is a TF which activates p21, p21 binds to cyclin E / CDK 2 and inhibits it
now that CDK 2 is inhibited, it will not deactivate pRb → which can bind E2F → now that E2F is inactivated; DNA synthesis enzymes are not expressed and the cell is arrested in G1 phase until the DNA is repaired
p53 also activates puma, puma inhibits BCL-2 which is anti-apoptotic, thus the cell is unable to prevent apoptosis
13. Molecular processes involved in the initiation and progression of cancer w. special respect to the properties and functions of the participating biomolecules
Cancer
is a group of diseases characterized by uncontrolled growth and spread of abnormal cells
tumor initiation and progression is a multistep process
several factors are involved in triggering and maintaining the pathophysiological state
Initiation and progression of neoplasia
cancer initiation is a multistep process
multiple genetic changes
activation of (proto) oncogenes and inactivation of tumor supressor
genes are all involved in initiation of tumors
Hallmarks of tumors
sustaining proliferative signalling
evading growth suppressors
activating invasion and metastasis
enabling replication immortality
inducing angiogenesis
resisting cell death
Clonal selection and tumor heterogenity
imagine a normal cell gets a mutation, allowing it to increase proloferation
afterwards the mutated cell clone will be mutated again, making it even more proliferative → this cycle continues
in the end, there is a heterogenous population of tumor cells with different genome
this is a problem in therapy agent tumors
External factors may lead to cancer
carcinogens are compounds, which may lead to the formation of tumors
chemical compounds: alfatoxins, benzopyrene, dimethylnitrosamine
radiation (⍺-radiation, UV-radiation
affecting tumor promoters: phorbol esters, hormones (e.g. estradiol)
some viruses may cause cancer
HPV: cervical carcinoma
Herpes: Burkitt’s lymphoma
Hep. B: liver cancer
Oncogenes
proto-oncogenes are genes for normal, physiological proteins in the cell, but have the potential to cause cancer if they are mutated
if mutated in a way they cause cancer, we call them oncogenes
the mutation that cause the transform from proto-oncogen to oncogenes are gain-of-function mutations
gain-of-function mutations are dominant, meaning that only one of the allele has to be mutated
ErbB
a mutation in EGFR gene, which is the receptor for EGF cause expression of ErbB
this receptor misses the binding site, and its tyrosine kinase activity is always active
this constant activation cause a constant “start proliferation” signal, even with no EGF binding
this mutated gene is caused by deletion
Ras
Ras is a G-protein, small GTPase, involved in cell signaling (insulin aswell)
a point mutation in RAS gene leads to an AA change from glycine to valine
the mutated Ros will lose its GTPase activity, meaning it is never inactivated
the loss of inactivation, is a gain-of-function and it will continously serine/threonine kinases (Ros, MEK, ERK)
Chromosomal fusion - philadelphia chromosome
reciprocal translocation of Chr 9 and 22 leads to the formation of the Philadelphia chromosome (shorter version of chr. 22) with a fusion gene “Bar/c-abl”
this fusion gene coes for Tel/PDGF receptor, a receptor tyrosine kinase which dimerizes even without binding PDGF → this activation leads to overactive proliferation
Tumor suppressors
mutations in tumor suppressors genes can cause cancer
tumor suppressor proteins are proteins that ensure that the cell doesn’t replicate unless its supposed to and only if there is no DNA damage
tumor suppressors either:
negatively regulate cell division
promotes cell death
participate in DNA repair
a mutation in tumor suppressors cause a loss-of-function mutation
recessive, meaning that both alleles must be mutated in order for cancer to proceed
Retina blastoma
retina blastoma protein is a protein which bind to E2F and inhibit it from acting as a TF for enzymes for DNA synthesis and promote passage from G1-S-phase
when phosphorylated by cyclin E / CDK 2 the pRb release the E2F, in case of loss-of-function mutation in pRb, the E2F is never inhibited, and the cells ability to proliferate incr.
p53
double strand break will lead to binding of MRN, this activates ATM and ATR which activates p53
p53 will incr. the expression of p21 which binds to CDK 2/ Cyclin E and inhibits it
a mutation in p53 will lead to cell division, even if there is DNA damage
PTEN
is involved in turing off the survival signal of the cell (PIP2 → PIP3 → AKT → BAD → apoptosis)
it PTEN act. is lost, the AKT will continuously signal for cell survival
14. Bioactive heterocyclic organic molecules: characteristic structural features, functional groups, biochemical-physiological role
Basics
they are cyclic compounds with one or more heteroatoms - different from carbon, mostly N, O or S
the majority of natural occuring compounds contain heterocyclic rings
we classify them based on the size of the ring and subdivided according to the quality of the heteroatom:
oxygen containing heterocycles → Furan
nitrogen containing heterocycles → e.g. Pyridine
sulphur containing heterocycles → e.g. Thiophene
they are also classified according to the character of the ring
Aromatic → e.g pyridine (double bonds within the ring)
Non-aromatic → Piperdine (no double bonds within the ring)
mostly common names are used
numbering starts at the heteroatom, while keeping other heteroatoms low
Fundamental properties of heterocyclic compounds
Electron distribution
4n + 2 delocalized p-electrons and/or lone electron pairs : aromatic
otherwise: non-aromatic
Geometry
aromatic → planar
non-aromatic → non-planar
Basicity
If lone electron pairs of N- are NOT involved in the aromatic system → Basic
If lone electron pairs of N- are involved in the aromatic system → not basic
Five membered heterocycles
Furan
a heteroaromatic compound of planar geometry
Thiophene
heteroaromatic compound
Tetrahydrofuran
a non-aromatic compound, a cyclic ether
it is the skeletal compound of furanose sugars
Pyrrole
heteroaromatic compound
does not have basic properties as the lone electrons contributes to the ring structure
it is a very weak base, can be deprotonated by potassium metal
Indole
a indole consist of a pyrrole + a benzene ring
tryptophan is a indole containing compound
it is an essential AA and precursor for serotonin
serotonin is a neurotransmitter in the GI-tract, it affects BP and insulin secretion
Pyrrolidine
The saturated derviative of pyrrole
pyrrolidine is non-aromatic
it is a cyclic secondary amine behaving as a base
the amino acids proline and hydroxyproline contains pyrrolidine skeleton
Imidazole
heteroaromatic compound
it is amphotheric → an pyrrole type N-H (very weak acid) and a tertiary amine (weak base)
histidine contains a imidazole ring
histidine is the precursor for histamine, important in allergic reactions
6 membered heterocycles
Tetrahydropyrane
a non-polar, non-aromatic compund
it is the skeleton for pyranose sugars
Chromane and Chromene
they are partially saturated skeletal compounds belonging to fused oxygen heterocycles
they occur in vit.E and the class of flavonoids
Flavonoids
secondary plant metabolites - plant pigments
they are great oxidants and complexing agents
bio-role in plants: protect chloroplast against infection and injury
protection against UV, attraction of pollinating insects
humans take up flavonoids in diet
Cathechin is similar, but with a chromane ring
Pyridine
heteroaromatic compound with N
also able to form H-bonds, it acts as an acceptor
colorless gas with unpleasant odor
soluble in water and good polar solvent
TOXIC
weak base
Pyridine derivatives
Vit. B6
Nicotine
Nicotine amide
Piperdine
reduction of pyridine yields piperdine
stronger base than pyridine
morphine is a piperdine derivative
Fused pyridine derivatives
Quinoline and isoquinolone
Pyrimidine
heteroaromatic planar molecule
acts as a weak base
similar properties to pyridine
essential compound in pyrimidine bases of DNA and RNA V
vitamin B1 (TPP)
Pteridine
fused heterocycle composed of a pyrimidine and pyrazine ring
component of folic acid and vitamin B2
Purine
The purine ring system consists of a pyrimidine and an imidazole ring
amphoteric compound
derivatives are purine bases (G and A) and caffeine / theobromine
Purine alkaloids
they are derivatives of Xanthine
stimulants of the CNS
increase BP, diuretic
caffeine → weak base
theobromine → amphoteric
Porphines
does not occur in nature → exclusively of theoretical interest, but has “daughter” structures that exist in nature
planar aromatic ring structurally related to 18 annulenes
porphyrines are macrocyclic natural compounds containing pyrrole rings
these compounds play a vital role in nature in oxygen transport in photosynthesis (clorophyll), in electron transport (cytochromes), vitamin B12
Vitamin B12
A porphyrin derivative
it contain a smaller macrocycle (corrin skeleton)
it is a Co⁺⁺⁺ complex with octahedral geometry
biological role: coenzyme (methylation, isomerase), methylmalonyl-CoA mutase and methionine synthase
Hem
Fe⁺⁺ protoporphyrin IX complex
important structural and functional unit of hemoglobin
it acts a a chelator for iron(II)ions
in the complex ion of octahedral geometry the four N-atoms donate one electron pair
cytochromes are heme containing proteins
the heme macrocycle is linked to the protein via a side chain (cysteine), in addition the iron ion is bound to the sulphur of methionine residue and the nitrogen atom of histidine
function: it’s redoxactive; Fe⁺⁺ ⇋ Fe⁺⁺⁺ + e⁻
important in electron transport
Bile pigments
the heme is broken down in a complex way
the intermediates of this process is biliverdin and bilirubin
biliverdin → green solid
bilirubin → yellow solid of lower solubility
bile pigments solubilization occurs in liver by glucuronation
jaundice is caused by increased levels of bilirubin in blood
Chlorophyll
modified porphyrin ring system
partially saturated pyrrole ring with cyclopentane ring, also two ester groups in the molecule
Mg⁺⁺ complex
function in photosynthesis
15. Vitamines. Classification, the biochemical role of the cofactors derived from them, with examples
Vitamines
vitamines are organic compounds which are necessary for life, and which the human body can’t synthetize (except vit. D and niacin)
many are cofactors in biological reactions
Water vs. lipid soluble vitamines
Lipid soluble
hydrophobic
can be absorbed efficiently only when there is normal fat absorption
transported in blood by lipoproteins or attached to specific proteins
diverse function
D, E, K and A (DEKA)
Water soluble
hydrophilic
mainly functions as co-factors
B, C
Vitamin B
Vitamin B1 → Thiamine
plays an important role in carbohydrate metabolism
it is phosphorylated to become TPP (thiamine diphosphate), an important cofactor
TPP is a cofactor for oxidative decarboxylation reactions and is needed for all 3 dehydrogenase complexes:
PDC
⍺-ketogluterate dehydrogenase complex
branched ⍺-keto acid dehydrogenase complex
(transketolase)
deficiency can cause Beri-Beri, Wernicke-Korsakoff and lactic acidosis
Vitamin B2 → Riboflavin
has a central role in energy-yielding metabolism
it is involved in redox reactions
it is converted into two prosthetic groups, FAD and FMN
FAD is needed for:
Acyl-Coa DH
PDC, and the other DH complexes
Succinate DH
Glycerol-3-P DH (mitochondrial)
FMN is needed in:
NADH dehydrogenase / complex 1
sources of riboflavin are milk and dairy products, meat, liver
Vitamin B3 → Niacin
Not strictly a vitamin, can be synthetized form the essential AA tryptophan
it is converted into NAD and NADP, which are involved in redox reactions
(nicotine amide ring)
it also regulates IC Ca⁺⁺ levels
NAD is also the source of ADP-ribose in ADP-ribosylation of proteins
Niacin def. causes pellagria, a photosensitive inflammation of skin
high levels of niacin is toxic
Vitamin B5 → Panthothenate
Panthothenate is a part of ACP and CoA
Vitamin B6 → Pyridoxine
is converted to PLP (pyridoxal phosphate)
PLP is an important cofactor for AA metabolism, especially in transamination
Glycogen phosphorylase
ALAT / ASAT
serine dehydratase
cystathione β-synthase
tyrosine aminotransferase
glutamate decarboxylase
histidine decarboxylase
steroid hormone action: removes the hormone - receptor complex from DNA binding → inactivation of the hormone
def.: due to increased hormone sensitivity to steroid hormone action it may be important in development of hormone dependent cancer
Vitamin B9 → Folic acid
converted to THF, a cofactor in reactions that move one carbon segments
THF is needed for:
GLycine cleavage enzyme
methionine synthase
serine hydroxymethyl transferase
Vitamin B12 → Cobalmin
cofactor in one carbon segment transfers, like folic acid
a vit. B12 def. leads to folic acid def. as B12 is needed for folic acid metabolism
B12 is needed for:
methylmalonyl-CoA mutase
methionine synthetase
Vitamin C
aka ascorbic acid, is a cofactor for proline and lysine hydroxylases
vit. C is also an antioxidant, and it incr. absorption of iron
vit. C is needed for:
Proline hydroxylase: collagen synthesis and HIF-1⍺
Lysine hydroxylase: collagen synthesis
vit. C def. causes scurvy, a condition caused by deficient collagen synthesis
impaired wound healing, loss of dental cementum
first islated by hungarian scientist Albert Szent-györgi
Vitamin A - Retinoic acid
retinal in vision
retinoic acid acts as a hormone binding to RXR which forms dimers with PPAR-𝛾
retinoic acid also has a role in regulation of gene expression and tissue differentiation
Two families of nuclear retinoid receptors
retinoic acid receptor (RAR): binds all-trans retinoic acid or 9-cis retinoic acid
retinoid x receptor (RXR): binds 9-cis retinoic acid. forms heterodimers with vit. D, thryoid and other nuclear receptors
Vitamin A deficiency
most important preventable cause of blindness
earliest sign is loss of sensitivity towards green light, followed by impairment to adapt to dim light, then night blindness
prolonged def. leads to xerophtalmia - keratinization of cornea and blindness
role in differentiation of immune system
even mild def. increase susceptibility to infectious diseases
Vitamin A is toxic in excess
Unbound vit. A cause damage to:
CNS: headache, nausea, anorexia → all associated with incr. CSF pressure
Liver: hepatomegaly, hyperlipididemia
Ca⁺⁺ homeostasis: thickening of long bones, hypercalcemia, calcification of soft tissues
skin: dryness, desquamination, alopecia (bald spots)
Vitamin D - cholecalciferol
can be synthesized from cholesterol in the skin upon UV-rad
cholecalciferol (vit. D3) can be converted to calcitriol after hydroxylation in the kidney and liver, calcitriol is the active metabolite
The principal function of vit. D is to maintain blood plasma calcium conc.
it incr. intestinal absorption of calcium
it reduces excretion of calcium, by stimulating reabsorption in DCT
mobilizes bone mineral
also involved in insulin secretion, synthesis and secretion of parathyroid hormone and thyroid hormone
Vit. D def.:
Rickets: bones of children are under mineralized as a result of poor absorption of calcium
osteomalacia: in adults from demineralization of bone
especially in women that is exposed to little sunlight
Vitamin D is toxic in excess
some infants are sensitive to intake of vit. D → leads to elevated Ca⁺⁺ in plasma → contraction of blood vessels, calcinosis-calcification of soft tissues
Vitamin E
does not have a precisely defined metabolic function
acts as a lipid-soluble anti-oxidant in the cell membranes
indirect inhibitor of PKC-regulation of smooth muscle cell growth
inhibitor of thrombocyte aggregation
premature newborns have less vit. E
RBC membrane is abnormally fragile as a result of peroxidation → haemolytic anemia
Vitamin K
is a cofactor for post-translational carbocylation of glutamate residues on proteins
proteins involved in blood clotting are dependent on this modification
gamma-carboxylation of (VII, IX, II, X) chelates calcium ions, allowing them to bind to membrane
def.: the clotting function is decreased
16. Molecular processes involved in iron metabolism with special respect to the properties and function of participating biomulecules
Iron
iron is an essential molecule in living organisms
it is involved in transport and storage of oxygen (heme) and is an integral part of many enzymes (aconitase, catalase, myeloperoxidase, complexes)
iron in free from is toxic, so it should be bound and its levels should be regulated
excess free iron goes through the fenton reaction (Fe⁺⁺ + H2O2 → Fe⁺⁺⁺ + OH⁻ + OH⋅) forming hydroxyl radical (ROS)
the body iron content is ∼3-4g
Iron homeostasis is mainly regulated at level of intestinal absorption (hormones may influence it)
Metabolically active iron
hemoglobin
“serum” iron, bound to a protein transferrin in blood
tissue iron: in cytochromes and enzymes
myoglobin
Storage iron
ferritin: found in blood, tissue fluids and cells
hemosiderin: found in macrophages and assessed by staining bone marrow w/ prussian blue
pathological form
Absorption of dietary iron
The absorption of iron takes place in the duodenum but begins in the stomach
gastric acid keeps the iron soluble and in the ferrous (Fe⁺⁺) form, as it is easier to absorb than ferric (Fe⁺⁺⁺) iron
dietary iron can either be inorganic, in free form, or as part of the heme, which is easier to absorb
if a Fe⁺⁺⁺ reaches the duodenum, it is reduced by duodenal cytochrome B → Fe⁺⁺ is then absorbed through a DMT1 (divalent metal transporter), which also absorbs zinc, lead and copper
heme is transported through a heme transporter
free iron is toxic to the cells, as they produce ROS through the fenton reaction, they are therefore stored in a protein called ferritin
one molecule can store up to 4,500 iron atoms
ferritin is mostly found in the liver
has ferroxidase act. ( Fe2+ → Fe3+)
two proteins, ferroprotin 1 and hephaestin, work together to transport the iron form the enterocyte to the blood
hephaestin is a ferroxidase which oxidizes Fe⁺⁺ to Fe⁺⁺⁺
ferroportin 1 transports the iron out of the cell
in the blood, Fe⁺⁺⁺ binds to apotransferrin (transferrin while bound to iron)
transferrin: synthetized by liver, localyy testes and CNS, binds 2 irons
ferroportin: on basal portion of syncytiotrophoblast, duodenal enterocytes, macrophages and hepatocytes
transferrin binds to transferrin receptors on the target cells.
most cells in the body express this receptor, but the erythrocyte precursors, hepatocytes and placental cells has highest density
when transferrin binds to its receptor, the Tf-TfR complex is transported into the cell as a endosome, containing both transferrin and the receptor
the pH is decreased by proton pumps, so the iron can dissociate from transferrin and escape into the cell, via DMT1
the only physiological excretion of iron is by shedding of epithelial cells in intestine and menstrual bleeding
Iron regulates the synthesis of its own key transport and storage molecules
iron metabolism is regulated at the expressional level by iron response elements (IRE) and iron response element binding protein (IRP), which can bind to IREs
the mRNA of both apoferritin and transferrin receptors contain IREs
when iron levels are low, IRPs are activated → they bind to IREs on both apoferritin and transferrin IREs
they bind to IREs on both apoferritin and transferrin IREs, the binding blocks translation of apoferritin, while prevent nucleosidases attacking mRNA of transferrin, thus activating translation
Low levels of iron:
IRP bind to mRNA of transferrin → Transferrin ↑ → iron transport into cell ↑ → cellular iron ↑
IRP bind to mRNA of apoferritin → Apoferritin ↓ → iron storage ↓ → cellular iron ↑
Hepcidin
the main regulator of iron absorption in the 25AA peptide hormone hepcidin
this protein forms complexes with ferroportin, which is then translocated into the cell and degraded
a high level of hepcidin will decrease the amount of iron absorbed
several factors influence hepcidin production
incr. iron levels incr. its production, as a negative feedback loop
its production is downregulated in hypoxia, anaemia and erythropoesis
during inflammation and infection, IL-6 induce hepcidin by STAT-3 dependent transcriptional activation
Iron def.
the most common cause for anemia
iron def./anemia in healthy males may indicate blood loss from tumor
Stage 1
depleted iron stores
asymptomatic
low ferritin
absent bone marrow iron
Stage 2
latent iron def.
low transferrin saturation
low serum iron
raised serum transferrin
normal hemoglobin
Stage 3
iron def. anemia
low hemoglobin
low hematocrit
Pica
the urge to eat non-food substances, like chalk, clay, soil, soap, paper…
often present in growing infants due to growth spurt
can occur after:
blood loss (period / hemorrhage)
malabsorption
poor intake
increased demand
Plummer vision syndrome
iron def. anemia dysphagia
atrophic glossitis
esophageal webs
middle aged women
can lead to tumor → treat by iron supplementation
Laboratory diagnosis
microcytic hypochromic anemia
decreased serum ferritin
decreased serum iron, decreased transferrin saturation
Causes of iron def.
physiological: menstrual bleeding, pregnancy, bleeding, growth
pathological: blood loss from GI, hemoglobinuria, malabsorption, colon cancer in males, diet, worms (parasites)
Principles of treatment
treatment of cause
iron replacement (oral, parenteral, blood transfusions)
Effects of iron overload
>10g iron
capacity of transferrin is exceeded → free iron in plasma → Fenton reaction → cardiac failure, cirrhosis, HSC senesence, DM, infertility
17. The biochemical role of macrominerals and trace elements, the molecular processes involving them. The molecular processes involving them. The molecular background of deficiencies and intoxications occuring at their abnormal concentrations
Trace elements
trace elements are chemical elements which should be available in the body in small amounts
often they are part of vital enzymes
as the name suggests, they should be present in traces, small concentrations. above a certain treshold, they become toxic for our body
the clue is to have them in a high enough quantity so that our body functions properly, but lower than the toxicity treshold
trace elements include: iron, manganese, copper, iodine, zinc, cobolt, fluoride and selenium
Macrominerals
needed in high amounts
they include: calcium, phosphate, mangnesium, sodium, potassium, chloride and sulfur
Iron
iron is essential for all living organisms
we req. 1-3mg of iron everyday
iron is absorbed as Fe2+ ions but stored as Fe3+ ions
many proteins contain iron, they can be divided into two groups:
the ones that contain iron in a heme group: hemoglobin, cytochromes, NO synthase
the ones that does not contain heme: Transferrin and ferritin
iron-sulphur proteins: complex 1 (NADH DH), complex 2 and 3
most notably iron def. leads to anemia
too high levels of iron leads to formation of hydroxyl radical via fenton reaction
Copper
transported in blood by ceruloplasmin and albumin
it is needed for some redox enzymes, like complex 4, superoxide dismutase and some hydroxylases (dopamine β-hydroxylase)
wilsons disease is a disease in which excess copper builds up in the body, in the liver, brain and kidney
there is low serum Cu2+ and ceruloplasmin and high urinary copper
copper is removed by complex formation with pencillamine
to prevent accumulation: a low Cu and high Zn diet
menkes syndrome is characterized by low cellular Cu uptake due to def. of ATP 7A membrane transport
good copper sources are seafood and liver, nuts, olives
Zinc
a trace element and needed for NO redox reaction
it is also important for DNA and RNA polymerase
it is located in the active center of enzymes like alcohol dehydrogenase, it assists selecting the right (-OH) amino acid for coupling to tRNA in thr-tRNA synthetase
it is found in metalloproteases, proteases, carbonic anhydrase
zinc is an also important part of zinc-fingers, which are binding domains for DNA
it is involved in olfaction, zn-def. → anosmia
zinc is relatively non-toxic, although toxicity symptoms like nausea, vomiting, epigastric pain and fatigue can occur in high doses of zinc
Chromium
Chromium is found in several forms Cr (III) and Cr (IV)
Cr (III) is thought to be a glucose tolerance factor, it increases glc-uptake in presence of insulin, decreases serum cholesterol in an unknown mechanism
Cr (IV) is toxic and carcinogenic
Avg. daily uptake: 50-200ug
Mangansese
A cofactor in oxidoreductases (xanthine oxidase), transferases, hydroxylases, lyases, isomerases, ligases, integrins, lectin, pyruvate carboxylase, arginase
It is a part of PP2A and glycogenin
Retroviruses (HIV) needs it, integrins
occupational exposure may lead to nervous system damage
def. cause coagulapathy and dermatitis
Molybedum
cofactor for xanthine oxidase
also needed for nitrogen fixation
Cadmium and mercury
mercury and cadmium has no physiological functions
they are “soft” heavy metals with very high affinity towards sulfur → they inhibit -SH enzymes
they can inhibit important enzymes like Ach-esterase, making them deadly
Aluminium
not necessary
not absorbed in considerate amounts
Al(OH)3 - aluminum hydroxide is used as antiacid
Lead
inhibits the first step (porphobilinogen synthase) of heme synthesis, disturbing any protein or enzyme needing heme
Arsenate
inhibits PPC and ⍺-ketogluterate DH
can also be incorporated in glycolysis instead of Pi, skipping the ATP-producing steps
can be treated by dimercaptopropranol?
Selenium
an important component of selenocysteine, an aminoacid based on cysteine that is used to build up slenoproteins, like glutathione peroxidase and deiodonases (T4→T3)
also found in muscles and sperm
it is possibly toxic, more reactive than sulphur
Fluoride
strengthen the enamel and protects against caries
best absorbed through water
RDA: 1mg/day
Iodide
essential for proper function of thyroid gland, production of thyroid hormones (T3,T4)
iodide def. causes goiter, enlarged thyroid gland
table salt usually added iodide, as iodate
18. The biochemical background and consequences of alcohol consumption
Alcohol
ethanol is the alcohol we drink, and it is poisonous to the body
the effects we associate with being drunk is actually a result of either the toxic impact or the body’s method of coping with the toxin
since it is toxic, the body immediately tries to eliminate it
alcohol is a drug and may cause dependency and addiction
alcohol is the primary cause of liver disease, it is toxic to GI, brain and pancreas
long-term abuse may cause nutrient def.
moderate amounts of alcohol, or at least alcohol containing drinks like wine, may have a protective effect agains cardiovascular diseases by increasing HDLs and decrease platelet aggregation → this may be due to polyphenols in the red wine
how much is one drink unit: depends on alcohol content, 1 glass of beer (300ml), 150ml wine, 2cl vodka (4%)
Route of alcohol
1. Mouth and esophagus
alcohol is diluted by saliva before being swallowed
some is immediately absorbed, especially highly carbonated drinks, such as champagne
2. Stomach
more alcohol is absorbed here, irritating the stomach lining of the stomach, increasing the acidity → acid reflux ?
males have alcohol DH in the stomach lining, females do not
3. Small intestine
any remaining alcohol is absorbed here
most alcohol is absorbed here
4. Blood stream
alcohol quickly diffuses through the body, affecting most cells
5. Brain
the cells in the brain are more suseptible because they are usually protected from toxins by the BBB
6. Liver
Blood-alcohol is metabolized in 2 stages and then respired into CO2, H2O and FAs
7. Excretion via urine, sweat and lungs
Processing
alcohol is processed in the liver, as it arrives with the bloodstream
there is no regulation of alcohol breakdown, other than availability of substrate
the 2 enzymes that metabolize alcohol are alcohol dehydrogenase and acetaldehyde dehydrogenase
catalase and cytochrome p450 can also convert ethanol into acetaldehyde
ethanol is first converted to acetaldehyde and then to acetate (acetyl-CoA)
the first reaction can be catalyzed by either alcohol DH, cytochrome p450 (specifically Cyp2E1) or catalase
ADH - cytosolic
CYP2E1 - ER/microsome
Catalase - peroxisomal
oxidation of ethanol to acetaldehyde, and then to acetate reduces NAD⁺ to NADPH, which increases NADH/NAD⁺ ratio in the cell, this increased ratio is not good, as NADH allosterically inhibits many reactions of the catabolic pathways
the cell then tries to lower the NADH/NAD⁺ ratio by using lactate DH and produce lactate → this leads to lactic acidosis
the “activation” of lactate DH also depletes the cellular pyruvate, an important substrate for gluconeogenesis, so the cell can not produce glucose, therefore alcohol intake may lead to hypoglycemia and lactic acidosis
the increased lactate in blood decreases the ability to excrete uric acid and may lead to Gout
the “stoppage” of TCA cycle and gluconeogenesis, increase ketogenesis and ketoacidosis forms
NAD⁺ is needed for β-oxidation of FA
the decrease of FA oxidation may lead to fatty liver and also increased FA synthesis
Cytochrome p450 / CYP2E1
catalyzes the formation of acetaldehyde from ethanol, in this reaction NADPH + H⁺ is needed
the oxidation of NADPH to NADP⁺ is not good → decreased ROS protection
CYP2E1 is inducible, meaning that alcohol can increase its function, it is also involved in the oxidation of many xenobiotics, e.g. paracetamol and propranolol
in case of prolonged alcohol abuse, CYP2E1 becomes overactive and metabolizes the drug too fast making it hard to correctly dose it
in case of hypertension, the β-adrenergic blocker propranolol may not have any effect, this overactivation of CYP2E1 may also increase ROS formation, which may increase toxicity of certain toxins
Acetaldehyde metabolism
acetaldehyde is metabolised by acetaldehyde dehydrogenase to acetate
acetaldehyde is toxic to the cell (even more than ethanol)
the balance between the various ADH and ALDH isoforms regulates the concentration of acetaldehyde, which is a key factor for the development of alcoholism
excess acetaldehyde can be caused by a decrease of ALDH activity → this induces an inflammatory response in the blood vessels → the face turns red
some asian populations have a decreased ALDH act. and are prone to flushing → asian flush
Long term effects of excessive alcohol consumption
Tissue damage
alcohol is an irritant to the mouth, throat and stomach
may raise the risk of cancer in these tissues
Liver damage
increased acetaldehyde levels in liver will stimulate inflammation in the blood vessels
ROS are formed and causes a fibrotic cancer
the fibrous scar tissue is formed by stellate cells → cirrhosis
Brain damage
alcohol can permanently damage brain cells, particularly in brains that are still maturing (brain finish development around 20-25 y/o)
Weight gain
alcohol has 7 kcal/g (carbs 4 kcal/g, FA 9 kcal/g)
19. The molecular source of oxidative stress, signal transduction pathways induced by oxidative stress, molecular tools and protection against oxidative stress
What is oxidative stress?
phenomenon caused by an imbalance between production/accumulation of reactive oxygen species (ROS) and the ability of the tissues to detoxify these reactive products
Reactive oxygen species (ROS)
free radicals contain unpaired, reactive free electrons
the free radicals are very reactive and are able to damage several biomolecules
some notable ROS are:
superoxide O2⁻⋅
hydroxyl radical OH⋅
hydrogen peroxide H2O2
peroxynitrite ONOO⁻
Radical: a molecule/atom with an unpaired valence electron, highly reactive
Some reactive nitrogen exists:
Nitric oxide - NO⋅
Nitric dioxide - NO2⋅
Nitrate radical - NO3⋅
Sources of reactive species
the sources of ROS and RNS may be endogenous or exogenous
examples of exogenous sources are exposure to pollutants, heavy metals, certain drugs, smoking, alcohol
the endogenous ROS are produced by both physiologically important enzymes and by non-enzymatic reactions
most notably, O2⁻⋅ can be formed in the reactions of the complex I and III in the resp. chain
semiquinone (⋅QH) is a radical and can easily pass an electron to O2 forming O2⋅⁻, so in this case the ROS is a by-product of the resp. chain
another enzyme forming a radical is NO-synthase, this enzyme is present in macrophage (oxidative burst), neurons and in endothelium (vasodilator), NOS catalyzes arginine to citrulline reaction by releasing NO
xanthine oxidase may produce radical oxygen species
NADPH oxidase → producing 2 O2⋅⁻
ROS can also be formed non-enzymatically, as in case of fenton reaction
excess free iron (in Fe⁺⁺ form) is toxic due to the increased ROS formation
Oxidative damage to..
Proteins
aggregation, fragmentation, cleavage
reaction with heme metal ion
modification of functional groups
→ change in enzymatic act. and ion transport
→ proteolysis
DNA
disintegration of the sugar ring
base modification
strand breaks
→ mutation
→ failure during translation
→ inhibition of protein synthesis
Lipids
saturation of unsaturated bonds
preparation of reactive metabolites (aldehydes)
→ changes in membrane fluidity and permeability
→ the membrane proteins are involved
Pathways induced by oxidative stress
oxidative stress can activate the NRF2 pathway. NRF2 is a TF which regulates expression of many genes related to protection of oxidative stress
in normal conditions NRF2 is bound to Keap 1, which inhibits NRF2. Keap 1 targest NRF 2 for ubiquitination and degradation
when there is oxidative stress, keap 1 will not inhibit NRF 2 and it translocates to the nucleus
in the nucleus it forms heterodimers with Mat proteins and binds to the ARE (antioxidant response element)
the mechanism of release from Keap1 is by oxidation of Cys residues
upon binding of ARE it induces expression of oxidative stress protection, like genes for NADPH synthesis, glutathione synthesis, or other genes for elimination of ROS
PARP
poly ADP-ribose polymerase is protein in DNA repair in physiological condition
in case of severe DNA damage, a lot of PAR is produced
this will deplete the cell of both NAD⁺, which is the source of ADP-ribose and ATP
less NAD⁺ = less glucose oxidation
cellular death is the result of PARP overactivation
ASK-1
apoptosis signal-regulating kinase 1
a MAPKKK (mitogen activated protein kinase kinase kinase)
when there is no oxidative stress it is bound to TRX (reduced thioredoxin) and is an oxidative stress sensor
oxidative stress releases TRX and TRAF (TNF-⍺ receptor associated factor 2) binds and ASK-1 is activated
ASK-1 phosphorylate other MAPKKK which phospphorylate JNK and p38 which leads to apoptosis
NF-kβ
ROS can also activate NF-kβ pathway
ROS activates IKKβ activates NF-kβ, which will induce the transcription of many pro-inflammatory cytokines, like TNF⍺, IL-1, IL-6, COX2 and iNOS
Cellular defense against oxidative stress
Antioxidants
compounds which functions to neutralize ROS
they can do this by accepting or donating electrons to eliminate the unpaired condition of the free radical, in this process they either directly react with the radical and destroys it, or become new, more stable radicals
examples: glutathione, vitamin A, C and E, uric acid
Enzymes
some enzymes has antioxidant effect, such as catalase, superoxide dismutase and glutathione peroxidase
20. Biochemical background of inflammation and septic shock
Basics
Inflammation is a biological response to damage or pathogens
causes can be burns, chemical irritants, pathogens, toxins, frostbite etc
Inflammation mechanism
Vasodilation
exudation - edema
emigration of cells
chemotaxis
phagocytosis
Systemic inflammatory response syndrome (SIRS)
at least two of the following symptoms:
temp. above 38 degrees or below 36
HR above 90
resp. rate >20
WBC above 12.000 or below 2.000
SIRS is not too high of x or too low, but deviation from the midpoint
Sepsis and septic shock
sepsis occur when the inflammatory reaction spread throughout the body
it is the 2nd most frequent cause of death in intensive care
mortality of 35%
septic shock has no effective cause or cure
some symptoms of septic shock are:
systemic vasodilation and hypotension
tachycardia
blood vessel endothel damage and edema: hypovolemia
insufficient blood flow to organs
intravascular coagulation
multiple organ failure
Biochemistry of inflammation
pathogens have specific markers on their surface → so called pathogen-associated molecular patterns (PAMPs) which are recognized by pattern recognition receptors (PPR) on the cells of the innate immune system, this binding will initiate a response
DAMPs are damage associated molecular patterns which are released by endogenous cells upon damage
e.g. the circular mtDNA is recognized as the circular bacterial DNA
in sepsis there is a cytokine storm, cells are damaged, releasing DAMPs activating even more immune cells, in a viscious cycle
some examples for PAMPs:
teichoic acid, peptidoglycan : gram + bacteria
lipopolysaccharide (LPS) : gram - bacteria
Toll-like receptors (TLR)
they are a family of PPRs the structure is a EC, N-terminal leucine rich repeat (LRRs), cysteine rich domains, transmembrane domain, IC Toll/ IL-1 receptor like domain
the different toll-like receptors recognize different molecules, but they have a common signalling pathway
binding of the PAMP will lead to activation of NF-kβ
NF-kβ consists of two subunits, p50 and Rel A
they are inactivated by IKB⍺, upon binding of a PAMP, IKK is activated
it is a kinase which phosphorylates IkB⍺ which is then ubiquitinated and degraded
NF-kβ can then go to the nucleus, bind to DNA and act as a TF for pro-inflammatory response
Organ failure - due to hypoperfusion
in the inflammatory response coagulation increase, while anticoagulation is decreased
one reason for this is that there is less activated protein C, thus there is no fibrinolysis and a thrombus occur → this leads to hypofusion
protein C is involved in many processes, so it has been a target in drug research, currently no drug is available
21. Proteins of the immune system, biochemistry of allergic reactions
Components of the immune system
innate immune system
response is non-specific
exposure leads to immediate maximal response
cell-mediated and humeral components
no immunological memory
found in nearly all forms of life
adaptive immune system
pathogen and antigen specific response
lag-time between exposure and maximal response
cell-mediated and humoral components
exposure leads to immunological memory
found only in jawed vertebrates
Innate immune system - TLR
toll-like receptors are essential receptors in the innate immune system and they function by recognizing PAMPs, like for example LPs (endotoxin/lipopolysaccharides), dsRNA, among other
the TLRs are consisting of 4 special domains
Leucine rich repeats (LRR)
Cystein rich region
transmembrane domain
TIR domain
not a kinase, but rather a docking station for proteins which will propagate the signalling
Cells of the innate immune system
NK cells
Mast cells
Phagocytes
Macrophages
Dendritic cells
𝛾δ T-cells
Adaptive immune system
Humoral immune response
consist of soluble immunoglobulins/antibodies which are secreted from the plasma cells
Cellular immune response
cytotoxic T-cells
MHC (major histocompatibility complex) presents internal proteins of cells, both self and foreign
specific receptor on the surface of T-cells (TCR) recognize MCH bound peptides
Immunoglobulins
they are proteins of the humoral immune response (adaptive immune response) which are produced by plasma cells
structurally immunoglobulins are heterotetramers of 2 light and 2 heavy chains
they have two “part” the Fc and Fab parts
the light chain occurs in two types: Kappa and Lambda, while heavy chain can be: ⍺, 𝛾, δ, 𝛆 and μ
the light and heavy chain are held together by disulfide bonds
smaller molecules can interact with specific AA residues while larger molecules interacts with all 6 AA loops in an induced fit
gene arrangement is responsible for the large diversity of immunoglobins produced
V-variable region and J-joining region can be changed while there is also an constant C region
Immunoglobulin G
most abundant Ig in the serum
consist of either k or 𝜆 light chain and 𝛾1-4 heavy chain
it exists in monomeric form (150kD) and is the only Ig which can pass through the placental barrier
when it binds to pathogen epitope it cause immobilization by agglutination, it also opsonize the pathogen, allowing the pathogen to be recognized and phagocytosed by macrophages and other phagocytic immune cells
IgG activates the classical pathway of the compliment system
it plaus an important role in antibody-dependent cell-mediated cytotoxicity and IC antibody-mediated proteolysis
associated with type II and III hypersensitivity
appears 24-48 hrs after antigen apperance
Immunglobulin A
the main antibody found in external secretion (saliva, tears, bronchial and intestinal mucous)
has a k or 𝜆 light chain and ⍺1 or ⍺2 heavy chain
appears in monomeric, dimeric and trimeric form, is secreted as a dimer
both IgA1 and IgA2 have been found in external secretion like colstrum, maternal milk, tears and saliva
IgA2 is most prominent in blood
causes degranulation of eosinophils and basophils
causes phagocytosis by monocytes, macrophages and neutrophils, and triggers respiratory burst activity by polymorphonuclear cells
Immunoglobulin M
first antibody to appear in the serum after exposure to an antigen
it has either a k or 𝜆 light chain and a μ heavy chain
it forms a pentamer and is the largest Ig (950kD)
spleen is the major site of production
it does not really diffuse and is found mostly in the blood
IgM are mainly responsible for agglutination of RBCs in case of non-compatible blood transfusion
Immunoglobulin E
only in mammals
consist either of k or 𝜆 light chain and 𝛆 heavy chain
occur in monomer form
plays an important role in type 1 hypersensitivity - allergic reactions
Immunoglobulin D
we know little about this, lowest quantity in sera
thought to have a role in allergy as it binds basophils and mast cells
Effector mechanisms of antibodies
Phagocytosis
IgG opsonization → recruits macrophages
Activation of complement system
can be activated by classical way (antigen-antibody), alternate pathway and lecitin pathway
it is a cascade where zymogens are activated to form a membrane attack complex
MHC 1
all nucleated cells express it
presents proteins endogenous of the cell
this can either be a peptide which is endogenous to our body or in case of virally infected cells it can be a viral peptide
consist of an ⍺ chain w/ ⍺1-3 subunits and a β-microglobulin
only the ⍺ chain is involved in peptide presentation
TCR
consist of ⍺ and β chains and are able to bind MHCI complexes
if there is a foreign peptide expressed on MHCI, cytotoxic T-cells will introduce perforin and granzymes onto the MHC I foreign peptide presenting cell, and kill it
MHC II
presents proteins bound to cell surface antibodies
the foreign peptide is internalized and proteolysed then expressed on MHC II
only APC cells have MHCII
consist of almost equal ⍺ and β chains, which are both involved in antigen
CD4+ T-helper cells recognize the MHC II
MHC II binds to the TCR and CD4
this causes the production of cytokines which activates other parts of the immune system
Hypersensitivity
abnormal response to antigens, 4 types
type 1 : anaphylactic reactions, IgE and IgG
type 2: cytotoxic, IgM and IgG
type 3: immune complex, IgG
type 4: cell mediated, T-cells
Type 1 : anaphylactic reactions, IgE and IgG
occur within minutes of exposure to antigen, antigen combines with IgE
IgE binds mast cells and basophils, causing them to undergo degranulation and relase mediators:
histamine: dilates and increases permeability of blood vessels (swelling and redness), increases mucous secretion (runny nose) and smooth muscle contraction (bronchi)
prostaglandin: contraction of smooth muscle of resp. system and increased mucus secretion
leukotrienes: bronchial spasm
anaphylactic shock: massive drop in BP, can be fatal in minutes
Mast cell mediators
Preformed:
vasoactive amines: Histamine
Neutral peptidases: tryptase, chymase
acid hydrolases: b-hexoaminidase
proteoglycans: heparin, chondroitin sulfate
adenosine (broncho constriction, platelet aggregation inhibition)
Newly formed:
eicosanoids: PGP2, LTC4
cytokines: TNF⍺, IL-4, IL-5, IL-6
Mast cell tryptase
tetrameric serine protease
found only in mast cells
Histamine
produced almost exclusively by mast cells and basophils
immediate pharmalogical effects
pruritus - itchy skin
vascular permeability ↑ / vasodilation (H1)
smooth muscle contr. (H1)
gastric acid secretion (H1)
FC𝛆RI - high affinity
found on:
mast cells
basophils
activated eosinophils
langerhans cells
Activation of mast cells
cluster of two or more IgE-bound FC𝛆RI by multivalent antigen
activation of protein tyrosine kinases
Lyn
Syk
transmission of signal
mediator release