Notes topic 9-

9. Post-translational modifications and IC targeting of proteins with special respect to the properties and functions of the participating biomolecules as well as the related characteristics of the participating cell organelles

Basics

• the product of translation: nascent poly peptide chain

  • often not ready to be a biologically active protein

  • modifications are often needed

Modifications on the N- and C-terminus

• The methionine is usually cleaved off, the N-terminus

• Amidation of the C-terminus

  • eg. vasopressin, oxytocin and cyclic peptides

Cleavage of signal sequences

• the signalling sequences are usually cleaved off once it enters its compartment

• one exception is nuclear proteins

Modification of individual aa?

• Phosphorylation - ser, thr, tyr

• methylation

• carboxylation

• sulfation - e.g. gastrin

• hydroxylation - pro, lys in collagen and HIF-1⍺

One example of post-translational modification is hydroxylation in collagen synthesis:

• in conversion of pre-pro-collagen to pro-collagen, some lysine and proline residues must be hydroxylated to create strong cross-striations in the finished product

• this hydroxylation requires vtit. C

• vit. C def. → scurvy ; bleeding gums due to dysfunctional collagen synthesis

Glycosylation, the attachment of carbohydrate side chains, is especially important in EC proteins, occurs in ER and golgi

Prenylation and farnesylation is typical in membrane anchoring proteins

• farnesyl and prenyl actas as anchors

Acetylation/deacetylation is very important in the case of histones, but also TFs and chaperones

ADP-ribosylation, catalysed by PARP in eukaryotes

Toxins causing ADP-ribosylation:

• Diphtheria toxin → ADP-ribosylation of EF-2 inhibiting it → cell death

• cholera toxin →ADP-ribosylation of Gs proteins → stimulates adenylyl cyclase → increased secretions of ions to GI → diarrhea

• pertussin toxin → ADP-ribosylation of Gi proteins → stimulates adenylyl cyclase → interrupts normal cell signalling pathways

Prostethic groups

some peptides needs other non-peptide units to function → these are prosethic groups

• hemoglobin → heme

• ferrition → iron

• alcohol DH → zinc

Proteolytic prosessing

some poly-peptide chains are inactive, and must have some parts cleaved off in order to be activated

• pro-enzymes or zymogens

Disulfide cross link formation

the formation of strong disulphide bonds may be necessary for proteins to protect their 3D-structure

this is especially important for EC proteins, as the EC environment is oxidizing, which would comprize the structures if not for these bonds

Protein targetting

proteins are synthesized on ribosomes in the cytosol, but other cell compartments needs proteins aswell

they are transported by the help of signal sequences, a short sequence of aa on the N-terminus (more common) or c-terminus (less common)

these sequences are recognized on the membrane of the target organelle. once inside the signal sequence is usually cleaved off

Transport to ER

• a protein called SRP binds to the signal sequence on the N-terminus - SRP consumes GTP during this process

• SRP then carries the ribosome-polypeptide complex, which has stopped translation, to the ER.

• SRP binds to SPR-R on the ER and resumes translation, which is now feeding the polypeptide into the ER

• Inside the ER, the signal is removed by signal peptidase

• Inside the ER the protein can be further modified, like with disulphide bonds or glycosylation

  • tunicamycin is an antibiotic which inhibits glycosylation.

• Following this, the protein travels from ER to golgi in transport vesicles in golgi proteins may be further modified

• after this golgi sorts the proteins, and sends them to their final destination

Protein targeting to the nucleus

• the signal sequence to the nucleus is not cleaved, the reason: nuclear envelope during cell division

• the signal sequence for proteins targeted to the nucleus in NLS, nuclear localization sequence

• it may be found anywhere on the peptide and not only on the termini

• a protein called importin is involved in the import of NLS tagged proteins. GTP is hydrolysed

Protein targeting to mitochondria

• protein targeted to mitochondria are fully synthesized in the cytosol, their signal sequences are recognized by proteins called TOM and TIM

• TOM 20, TOM 22, TOM 40 are involved in transport across the outer mitochondrial membrane

• TIM 22, TIM 23/17 and TIM 44 are involved in transport across the inner mitochondrial membrane

• after entry to mitochondria, the signal seq. is cleaved

Protein targeting to peroxisomes

• signal sequence PTS-1 on C-terminus or PTS-2 on N-terminus

  • PTS-1 is one of few C-terminal signal sequences (Ser-Lys-Leu)

• So called peroxins recognize the PTS signal and transport them into the peroxisome

Protein targeting to lysosomes

• the signal sequence for proteins targeted to the lysosome is mannose-6-P residue

• on the membrane of lysosome there are mannose-6-P receptors, which recognizes and binds the protein, which is then transported into the lysosome

Protein targeting of transmembrane proteins

• transmembrane proteins reach their target by containing special signal sequences called “internal stop-transfer anchor sequences” and “internal signal-anchor sequences”

• they are transported through the membrane, but at the STA and SA sequences it is embedded into the membrane

Receptor-mediated endocytosis

• receptor-mediated endocytosis is a process by which cells can take in EC proteins

• cells depend on external proteins to a large degree

• for cells to take up for example LDL, transferrin, peptide hormones or circulatory proteins from the outside, receptor-mediated endocytosis is needed

• there are 3 main pathways of receptor-mediated endocytosis

  • clathrin-dependent endocytosis (most common)

  • caveolin-dependent endocytosis

  • clathrin- and caveolin- independen endocytosis

• in clathrin-dependent endocytosis the EC protein in questions binds to receptors on the cell surface, the IC protein clathrin then binds to the inside of the cell membrane at the area where the EC protein is bound to its receptor

  • clathrin then forms an invagination in the cell membrane, first forming a pit and later a vesicle, containing the receptor and EC protein and internalises it

  • a protein called dynamin holds the clathrin-coated vesicle together

  • the EC protein dissociates form the receptor inside the vesicle, and the vesicle is split to two, one contains the receptor, which is moved back to the membrane, and the other w. the EC protein.

  • The protein can then be transported to where it is needed

12. Plasma proteins: classification, properties, biochemical-physiological role

Basics

• blood has several functions in the body → it transports O2 and nutrients, metabolic waste, hormones.

  • it also has regulatory functions: thermoregulation, pH reg. (buffer)

  • protective functions: hemostasis by activating platelets and initiating blood clots, prevents infection by antibodies and WBC

• blood has two major components:

  • liquid: plasma (55%)

  • formed elements (45%): RBC, WBC, platelets

• blood plasma

  • water (90-92%)

  • proteins (6-8%): albumin, globulins, fibrinogen

  • organic nutrients: Glc, carbs, aa’s

  • electrolytes: Na+, K+, Ca++, Cl-, HCO3-

  • non-protein nitrogenous substances: urea, creatine

  • respiratory gases

Families of blood proteins

Protein electrophoresis

Prealbumin

• named as prealbumin, as it migrates faster than albumin in classic electrophoresis

• aka. transthyretin

• it is the transport protein for thyroid hormones and Vit. A (retinol)

• prealbumin is decreased in:

  • liver disease

  • nephrotic syndrome

  • acute inflammatory response

  • malnutrition

Albumin

• family of water soluble proteins which are able to transport other molecules non-specifically

• it is the most abundant protein in the blood

• albumin is synthetized in the liver and has a half-life of ∽20 days, making it a good indicator for liver funtion

• it is the main constituent for oncotic pressure

• albumin should not be present in urine

  • this condition is called albuminuria and is mostly pathological, traces can be seem in pregnancy or after running marathons, but normally it should not be present in urine

• functions

  • oncotic pressure

    • albumin is responsible for ∽80% of the plasma oncotic pressure

    • hypoalbunemia leads to edema

  • buffering

  • transports

    • hormones, calcium, drugs (salicylates), FFA, bilirubin

• causes of hypoalbunemia

  • arteficial: diluted sample

  • physiological: pregnancy

  • decreased aa: reduced synthesis of non-essential or low dietary essential aa’s

  • increased catabolsim: surgery, trauma and infections

  • liver disease

  • inc. loss from kidney

Prothrombin

• a glycoprotein occuring in blood plasma and is an essential component of the blood-clotting mechanism

• prothrombin is converted into thrombin by clotting factor X or prothrombinase

  • thrombin then acts to transform fibrinogen to fibrin

• under normal conditions, prothrombin is changed into thrombin only when injury occurs to the tissue or circulatory system

• synthesis of thrombinogen requires vit. K, and hypothrombinemia causes prolonged bleeding

⍺1-antitrypsin

• a protein synthetized by liver and macrophages

• it is an acute-phase protein, meaning that it is produced upon a stress

• causing secretion of cytokines (IL-1, IL-6, TNF-⍺)

• it’s function is to inhibit proteases, this is due to bacterias and leukocytes can release proteases, especially elastase, which is breaking down elastin in the lungs

• in antitrypsin deficiency, 1aa can be wrong

  • liver produce antitrypsin, but can’t release it, this causes liver cirrhosis, neonatal jaundice and pulmonary emphysema in adults

Alpha-fetoprotein

• a glycoprotein which is the major plasma protein produced by the yolk sac and the liver during fetal development

• it is thought to be the fetal form of serum albumin

• AFP can bind copper, nickel, FA, bilirubin and is found in mono-, di- and trimeric form

• levels of AFP above 500ng/ml can be indications of hepatocellular carcinoma, germ cell tumors and metastatic cancers of liver

Positive acute phase protein

• inc. during stress/inflammation due to production of cytokines (IL-1, IL-6, TNF-⍺)

• these are:

  • ⍺1-antitrypsin

  • heptaglobulins

  • ceruloplasmin

  • fibrinogen

  • C-reactive protein (CRP)

C-reactive peptide

• is an acute phase protein synthetized by liver

• it precipitates the polysaccharide (fraction C) of pneumococcal cell walls

• it’s important for phagocytosis

• in inflammation CRP plasma levels increase

• in physiological cells it binds to phosphatidylcholine on apoptotic cells and activates the complements system

Ceruloplasmin

• synthetized by liver

• binds and transport serum copper

• it is important in acute phase response, as it can inactivate ROS and prevent tissue damage

• plasma levels is lower in Wilson’s disease in which copper is accumulating in liver leading to cirrhosis

Haptoglobin

• synthesized by liver

• it binds free hemoglobin to form complexes which are metabolized in the RES

• it limits iron loss, as Hgb is small enough to be filtered by glomeruli

• decrease: hemolysis

• increase: acute phase response

Finbrinogen

• synthetized in liver, is an acute phase protein

• its function is to form a fibrin clot, when activated by thrombin

• removed in the clotting process, not seen in serum

• its level increases during pregnancy and by using oral contraceptives

Alpha-2-macroglobulin

• synthetized by liver, macrophages, fibroblasts and adrenocortical cells

• is the largest major non-immunoglobulin protein in plasma

• acts as an antiprotease, supressing many proteases

• acts as a carrier protein, binds cytokines, GFs, insulin, TGF-β

Hypergammaglobulinemia

• increased immunoglobulin levels may be result of stimulation of many clones of β-cells (polyclonal hypergammaglobulinemia) or monoclonal differentiation (paraproteinemia)

Polyclonal hypergammaglobulinemia

• stimulation of many clones of β-cells produce a variety of antibodies that appear as diffuse increase of 𝛾-globulins on electrophoresis

• e.g. acute and chronic infections, auto-immunity

Monoclonal hyperproteinemia

• proliferation of a single B-cell clone

• produces a single antibody, which appears as a distinct densely stained line on electrophoresis (paraproteins or M band)

• paraproteins are characteristic of malignant B-cell proliferation

• multiple myeloma

14. Alterations of protein metabolism in various physiological and pathological conditions

Basic

• proteins are essential molecules for normal cellular functions while at the same time containing lots of amino acids, which can in low energy state be broken down to produce energy

Well-fed stage

• in the well fed state, there are surplus of energy in the body, meaning that we have sufficient amounts of ATP

• as we know, protein is heavily dependent on ATP/GTP to proceed

• the availability of AA’s, which are substrates of protein synthesis also increases the protein synthesis

• lastly, we know that insulin is a so-called anabolic hormone, this means that it promotes the synthesis of protein

• insulin also suppress the activity of gluconeogenesis by activating PFK-2

  • this reduces the need for gluconeogenetic substrates, which is exactly what the carbon skeleton of glucogenic amino acids yield

Fasting stage

• when it is long since the last meal, the glycogen storage has been depleted, and the body starts to search for alternate methods to produce glucose to feed the important organs, like the brain

• the AAs are good substrates for producing both glucogenic and ketogenic substrates, both which can fuel the brain

• proteins should be broken down in order to produce free AAs to fuel gluco- and ketogeesis

• it is important to notice than even though protein gets broken down, there is still production of proteins, but the type of proteins differ

• in pathological condition the acute phase proteins are typically increased in synthesis

Cortisol

• during stress response, cortisol is secreted from the adrenal gland

• cortisol has an catabolic effect on muscle tissue and cause breakdown of proteins into AA’s, so that they can be used as substrates for gluconeogenesis

Diabetes mellitus

• in DM our body enters a “pretend” starvation

• our body does not send (type 1) or receive (type 2) the insulin signal which is the body’s way to signal when we have a enough glc

• so processes in the body, starts to break down proteins in order to produce glc

• remember that insulin signalled to not break down proteins, so in this case the preventative signal is not sent, so proteins are broken down

Different alterations that shift protein metabolism

• in different pathological (and physiological) conditions some proteins will be more or less synthetized

Hsp

• heat shock protein are produced when the temp incr. or other environmental stress occur

• hypoxia can promote it

HIF-1⍺

• protein which in normal conditions are constantly hydroxylated and degraded, however in hypoxia, it will not be degraded

• it will then promote Cox 4-2, promote angiogenesis, and other hypoxic stress factors

15. Antibiotics

Basics

• antibiotics are molecules or substances produced to kill microorganisms

• more than half of them act agains protein synthesis, most of them act on the ribosomes

• the fact that it acts on the ribosomes makes it problematic, as the ribosomes are highly conserved, this it is hard to find a specific inhibitor which is not toxic to the host

Aminoglycosides (streptomycin)

• inhibit initiation and causes misreading of mRNA in bacteria (prokaryotes)

Tetracycline

• binds to the 30s subunit and inhibits the binding of aminoacyl-tRNA

• prokaryotes

Chloarmphenicol

• Inhibits the peptidyl transferase activity of 5os ribosomal subunit in prokaryotes

Cycloheximide

• inhibits translocation in eukaryotes

Erythromycin

• binds to 5os and inhibits translocation in prokaryotes

Puromycin

• causes premature chain termination by acting as an analogue of aminoacyl-tRNA in both eukaryotes and prokaryotes

Neomycin

• Inhibits binding of AA-tRNA to bacterial ribosome

Antibiotics that do not inhibit protein translation

Penicilin

• block formation of peptide cross links in peptidoglycans, weakening the bacterial cell wall

Antimycin

• inhibits complex III in the rep. chain

• inhibits oxidation of ubiquinol

Oligomycin

• Inhibits proton flow through F0 of F1/F0 complex

ABC-transporters importance in antibiotics resistance

• ABC-transporters are multidrug transporters that are able to export antibiotics out of the cell

β-lactamase in penicillin resistance

• penicillin and related antibiotics contain β-lactam rings, these can be destroyed by β-lactamase, thus making penicillin ineffective

• an idea could be to administer β-lactamase inhibitor with the antibiotics

17. Biochemical background of dysfunction associated with hemoglobin variants

Hemoglobin

• Hgb is a heterotetromer consisting of 2⍺ and 2β subunits in adult hgb

• heme is a porphyrin ring with central iron (Fe⁺⁺), iron is the site of O2 binding

Genes

• the chromosome 16 codes the ⍺-chains + ɸ chain

• the chromosome 11 codes the β, δ, 𝛾, 𝛆

Fetal development of Hgb

• in the first weeks the phi and 𝛆 are the dominant subunit

• after approx 3 week the ⍺ subunit is synthetised as well as the 𝛾

• after birth more and more β-subunits are produced

• why is this important → ⍺𝛾 Hgb has higher affinity towards O2 so the fetus can “steal” O2 from the maternal ⍺β Hgb

Hemoglobinopathies

• structurally abnormal Hgb is synthetised

• more than 1.000 exists, but ∼100 are associated with disease

• sickle cell disease (Hb S) and hemoglobin C have a similar structurally deficiency, where the 6th AA from the N-terminus is changed from glutamate to valine-Hb S or lysine-Hb C (negative AA → neutral or positive AA)

Sickle cell disease (Hb S)

• in sickle cell disease there is a point mutation in the gene coding for hemoglobin

• the point mutation leads to substitution of glutamate to valine in the 6th AA from the N-terminus

• Hb S polymerizes when deoxygenated and cause the sickle shape of the blood cells

• the deformation reduces its ability to circulate and will be able to cause infarction and hypoxia

• Hbs is usually diagnosed early in life, when Hb F levels decrease, patients have severe chronic anemia with several complications

  • clincial features incl.:

    • abnormal growth of bone and joints (hand-foot syndrome)

    • renal complications

    • spleen and liver (autosplenectomy, hepatomegaly and jaundice)

    • enlarged heart, pulmonary infarction

    • leg ulcers

    • infections - main cause of mortality

Hemoglobin C

• in the case of Hb C it is the same 6th N-terminal AA, glutamate, which is switched to lysine

• it is associated with splenomegaly, abdominal discomfort and hemolytic anemia

• diagnosed by Glod-ber formations in a blood smear

• most common in West-Africa

⍺-Thalassemia

• this is a quantitative Hb disorder

• absence of ⍺-chains will result in increase of 𝛾-chains in fetal life and β-chains in adults

• the severity will depends on how many of the 4 genes (2 per chromosome 16) are affected

  • silent carrier: aa/a-

  • minor: —/aa or a-/a-

  • Hg H: a-/—

  • Barts hydrops fetalis — / — (non compatible with life)

⍺-thalassemia trait (minor)

• aa/a- or —/aa

• exhibits mild microcytic /hypochromic anemia

• normal Hb electrophoresis, may be mistaken for iron def. anemia

Hemoglobin H disease

• second most severe form

• only one functioning gene for ⍺-chain

• excessed unpaired 𝛾 or β chain can form stable complexes which bind O2 with high affinity

• RBCs are microcytic, hypochronic

• lower Hb and Hb H can be oxidized and be percipitated for form heinz bodies

• GOLF apperance can be seen in blood smear with cresyl-blue stain

Bart hydrops fetalis syndrome

• most severe form, incompatible with life

• there are no functioning genes for ⍺-chain (—/—)

• babies born with hydropsis fetalis has acites and edema, hepato- and splenomegaly

• severe anemia and no normal RBC

β-Thalassemia

• usually caused by mutations on the 11th chromosome

  • unlike in ⍺-thalassemia, the excess ⍺-globin will not form complexes with high O2 affinity

β-thalassemia trait (minor)

• caused by heterozygous mutation

• usually presented with mild hemolytic anemia, although during stress it can increase

• Hgb levels are normal

• they have high HbA2 and normal/slightly elevated HbF levels

β-thalassemia intermedia

• there is an increase in HbA2 production and HbF

• anemia, jaundice, spleno- and hepatomegaly may occur

• significant increase in bilirubin levels

β-thalassemia major

• characterized by very severe microcytic, hypochromic anemia

• low levels of Hb (2-8 g/dl) - normal 14-18 g/dl

• bone changes due to expansion of bone marrow for erythropoesis

• no normal RBCs on blood smear

• have skull deformation, hepato- and splenomegaly + growth retardation

• requires transfusion

  • risk for AIDS

  • iron overload → chelation required to prevent cardiac problems

  • alloimmunization

18. Molecular processes affected and biomolecules participating in certain acquired disorders of protein metabolism with special respect to ER stress and neurodegenerative diseases

ER stress

• the ER is under constant stress but the degree of which can be influenced by external factors

• ER stress can be induced by inhibitors of protein glycosylation (physiologically by starvation, by tunicamycin)

• decrease of intraluminar Ca⁺⁺ conc. by inhibition of SERCA (sacroendoplasmatic reticulum calcium ATPase) or ionsphoresis

• intraluminar reducing agents and protein overproduction (e.g. viral infection)

Response

• the ER will respond by increasing the ER volume and functional capacity

• also it will decrease the protein overload but inhibiting protein synthesis and protein degradation

• worst case: apoptosis

Role of ER stress

• sensing of fuel molecule levels, conversions of β-cells to plasma cells and in biotransformation of exogenous enzyme in liver

Unfolded protein response (UPR)

• the ER stress is sensed by molecules that are embedded into the ER membrane (IRE1, PERK, AFT6)

• the IRE1 (serine/threonine - protein kinase /endoribonuclease ) forms homodimers

  • form complexes with BiP (Hsp) when there is excess of unfolded protein in the lumen

  • IRE is released from ER membrane and splices XDP-1 mRNA, allowing expression of XRP-1

• XRP-1 is a transcription factor for chaperones and proteins for upr, this leads to the early response of UPR

  • the early response increase volume of ER and expression of folding machinery. also ERAD (ER associated degradation of protein)

• in case of continued signalling after early response, the late response will be activated (via ATF-4 and chop) → apoptosis

• PERK (PKR (RNA-activated kinase) - like ER-kinase) is activated by BiP and phosphorylates eIF2⍺

  • this stops all protein synthesis except for ATF-4

  • ATF-4 is a TF for AA-import, GSH synthesis and CHOP

    • CHOP induced apoptosis

• PERK also also phosphorylates Nrf 2 which is an TF for oxidative defense, expressing anti-oxidants

ER overload response

If there is an overload of proteins (viral inf.) the ER overload response starts. This response increases inflammation

the 3 steps of this response is:

1. Ca⁺⁺ outflow from ER

2. ROS formation

3. Activation of NF-kβ → cytokine production

this response is associated with many diseases like: Alzheimers, CF and marfan syndrome

Cystic fibrosis

• disease of the lungs associated with misfolded CTRF (ABC-transporter), causing water retention and more viscous mucus

Neuodegenerative diseases

Demenita - revesible

• some forms of dementia can be reversed

• that can be in case of vitamin B12 and folate def,, hematomas etc

• dementia is not a disease, but rather a symptom that may be caused by several diseases and underlying factors

Irreversible dementia

• many dementias are not-reversible, such as: Alzheimer, Parkinsons, Huntingtons, Pich’s and Balo’s

• a common feature of dementias are atrophy of the brain → narrower gyri and wider sulci, due to death of neurons

Amyloids

• amyloids are aggregates of proteins that has been folded into a special shape that allows them to stick together and form fibrills

• These amyloids are usually comprised of β-sheets, and are sometimes called β-amyloids

• when proteins are clumped together like this, cells are damaged

• a disease where amyloid formation occur is a type of amyloidosis

• the specific protein that form amyloids vary from disease to disease

• in alzheimers amyloids are formed from fragments of APP(amyloid precursor protein)

• in parkinson, ⍺-synuclein form amyloids

Alzheimers disease

• alzheimers disease in an amyloidosis

• the fibrils formed are called Aβ-plaques and are caused by specific cleaving of APP by ⍺ or β-secretase

• in addition to being amyloidosis, alzheimer’s is also associated with formation of “neurofibrillary tangles”

• to musch phosphorylation of a microtubule stabilizing protein called TAU, is associated with many diseases, they are called taupathies

• the hyperphosphorylated TAU causes disintegration of the microtubules and TAU aggregates as neurofibrilar tangles

Parkinson’s disease

• parkinson’s is caused by loss of dopaminergic neurons in the substantia nigra → this leads to the loss of inhibition of striatum, through the nigrostriatal tract

  • as there is no inhibition, cells of the striatum dies from excitocytotoxicity

• many proteins and genes are associated with parkinson’s

  • under pathological conditions, a protein called ⍺-synuclein aggregates and forms amyloids, which eventually form lewy bodies, a classical histological marker for parkinson

  • dopamine stabilizes the oligomers of the lewy body formation

• PlNK-1 and LRRK2 mutation can inappropriatly phosphorylate proteins, which disturbs signalling and eventually leads to cell death

• DJ-1 mutation leads to proteasome inhibition, abnormal phosphorylation and oxidative stress → neuronal cell death

• Parkin, a ubiquitin ligase, mutation leads to less proteosomal degradation of misfolded proteins → accumulation of misfolded proteins → neuronal cell death

Parkin/PlNK-1 mediated mitophagy

• in normal conditions, mitochondria goes through fusion and fission

• it is essential for biogenesis and quality control of mitochondria

• the mitochondria which are unable to fuse are degraded by mitophagy

• for fusion, adequate membrane pot. is needed and leads to accumulation of mitofusion, a prot. which initiates fusion

  • mitofusion can be excessively phosphorylated by parkinin and PlNK

Huntington’s disease

• huntington is raised by a loss of neurons in the caudate nucleus

• when the gene that codes for a protein called huntingtin, has too many CAG sequences in the gene

• this occurs due to a special type of mutation called trinucleotide repeat expansion, which causes the trinucleotide CAG to repeat many times in a gene

• healthy people has less than 26 repeats, as the number of CAG repeats increase, the risk for disease increases

• CAG codes for glutamine

• the result is a huntingtin with too many glutamine residues, having a polyglutamine tract

• huntingtin normally increase SRE-regulated proteins

• they are proteins important for cholesterol synthesis in the CNS

Friedrich’s Ataxia

• chromosome 9

• is also a trinucleotide expansion disorder, where the GAA is repeated to many times in the Frataxin gene

  • this reduces expression of frataxin

• Frataxin is a protein involved in formation of iron-sulphur clusters and several proteins will lose function due to the frataxin mutation (succinate DH, ferrochetalase, aconitase..)

Prion related diseases

• prions are misfolded proteins which acts as chaperons, but they will cause misfolding of proteins, forming β-plated sheet which are depositing

• in humans, the most important protein is PrP which usually is in the c-conformation PrPc, however sometimes this folds into PrPsc form which is resistant to proteolysis and cannot be broken down

• when the PrPsc comes in contact with another PrPc it changes conformation to PrPsc

• many proteins in PrPsc form clump together and forms amyloids which causes neurodegradation

19. The composition and physicochemical characters of biofluids

Biofluids

• in the body there are several types of biofluids, one group can be considered as filtrate of blood

• they are in constant exchange of substances with blood, so the number of various solutes in them are in dynamic equilibrium with their conc. in blood

• the filtrates are: urine, saliva, CSF and synovial fluid

• Gastric juice, duodenal fluid, semen and amniotic fluid, as well as exudates and transudates (which are found in pathological conditions)

• Lymph is also an important biofluid

Blood plasma

• blood plasma contributes to ∽55% of total blood volume, and is a protein-salt solution which acts as a suspension for RBCs and WBCs

Composition

• 90% water

• 8% protein

  • albumin, globulins

• 0.9% Inorganic salts

  • Na⁺, K⁺, Ca⁺⁺, HCO3⁻ and PO43⁻

• 1.1% Organic substances

  • Glc, urea, uric acid

Urine

• the average amount of urine is 800-1500ml, it depends on liquid intake and other ways of liquid (sweat, stool, respiration)

• the color of urine normally falls between pale straw yellow to amber depending on its concentration

  • color can be influenced by solute conc., pathological molecules, drugs and diet

  • red color suggests blood or free Hgb is in urine (hemogloburia)

  • brown color - bilirubin

composition

• the chemical composition of urine is constantly changing and depending on diet

• conc. of solutes keep changing throughout the day

• dry weight of excreted materials = 60 g/day

• hormones can be found

  • hCG: pregnancy test

  • LH: ovulation test

Stuff that should not be in urine

• proteins → proteinuria

• glucose → DM / glucosuria

• RBCs → hematuria

• Hemoglobin → hemoglobinuria

Lymph

• a clear to white fluid made of WBCs and chyle (a fluid form the intestine)

• lymph is involved in many processes in the body

• chylomicrons will be transported from the intestines to the liver in lymphatics

• it is also involved in the immune system

Composition

• composition of lymph is roughly equal to that of blood plasma

Synovial fluid

• found within the synovial/joint capsule and is produced by the synovium

• it is a slippery fluid which lubricates the joint surfaces in order to reduce friction and pain

Composition

• proteoglycan - Lubricin

• GAGs - Hyaluronan

• proteins below 12kD due to non-selective plasma filtration → plasma content is roughly equal to plasma

Functions

• reduce friction

• shock absorption - non-newtonian character

• nutrient and waste transport from chondrocytes

20. Osmosis, osmolarity with contribution of different solutes

Osmosis

• in general, osmosis is the movement of the solvent from the higher place of solvent concentration, to a place of lower solvent concentration, through a semipermanent membrane

Osmotic pressure

• the pressure that is needed to counteract the flux of solvent, given by the formula: ∏ = icRT

  • ∏: osmotic pressure

  • i: van’t hoff factor

  • c: molarity of all dissolved substances

  • R: universal gas constant

  • T: temp in Kelvin

• the osmotic pressure is a colligative property of a solution, colligative means that the property depends only on the number of the dissolved particles but not on their type or chemical composition

• in living organism, osmosis is related to the movement of water across the cell membrane, it will flow from the place of higher water concentration to the place of lower water concentration

Osmolarity

• total number of all osmolytes (in moles) in 1L solution

Osmolality

• total number off all osmolytes (in moles) in 1kg solution

Osmolytes

• osmotically active dissolved particles (AAs, sugars, polyols, urea)

Blood plasma

• the osmolarity of blood plasma is 290 < Cbp < 310 milliosmoles

  • this equals a NaCl solution of ∽150 mM NaCl (0.9% NaCl) → physiological saline solution, this is isotonic

• if we were to place a blood cell in sterile water, which is hypotonic water will flow into the cell to counteract the osmotic difference → burst of RBC

• if we place and RBC in hypertonic solution, e.g 20% NaCl, the water will flow out of the cell and the RBC shrints

Importance of osmosis in humans

Blood plasma

• there is a regulation of BP in the kidneys, by water flow into urine

• ANP will decrease BP by increasing water in the urine, this is accomplished by increasing excretion of Na⁺ into the urine

  • Na⁺ is an osmolyte which “drags” water with it into the urine

Diarrhea

• in the case of osmolytes in high conc. in the intestinal lumen, like lactose due to lactase def.

• these is an increase of lactose, an osmolytic in the lumen, water is dragged into the intestine and watery stool is the result

Sense of thirst

• intiated by osmoreceptors cells outside the BBB

Edema

• albumin is an important osmolyte in blood plasma in order to keep plasma oncotic pressure

• in case of liver failure the oncotic pressure of plasma falls, filtration occur and water sieves out of the blood

• accumulation usually occur in legs due to gravity