Appunti di Patologia (corso OMP)

INTRA AND EXTRACELLULAR PROTEIN ACCUMULATION

This is an issue strictly related to aging.

Ex. Alzheimer syndrome (extracellular accumulation -> amyloidosis).

Protein synthesis can happen thanks to the ribosome associated to the Endoplasmic reticulum(ER). Inside the ER protein folding is regulated, here are performed protein quality controls to be sure that they are correctly folded and can properly work. If a protein is not properly folded it is recognized (multi-chaperone complex, HSP40 and HSP70) and transported outside the ER, in the cytosol the unfolded protein will be ubiquitinated and degraded in a proteasome dependent way (protein don’t accumulate in the ER or cytosol).

If the ER is producing misfolded proteins, it translocates them outside so they can go to degradation, but the main problem is to STOP the production of the unfolded protein, otherwise the system will go to saturation (the production should not overwhelm the degradation).

There are different stimuli that will

result in the production of unfolded/misfolded proteins, when they start to accumulate, the ER is in a STRESS condition. Frequently ER stress is induced by treatments-> drugs. The ER has an adaptative response that can be established with 2 different pathways:

The first one is the UNFOLDED PROTEIN RESPONSE (UPR), that acts in the bulk reduction of the production proteins. Chaperonin levels increase, some proteins are specifically overproduced because they are strictly required in specific action.

The second pathway is the ER ASSOCIATED PROTEIN DEGRADATION (ERAD), which is a stressed upregulated protein degradation (not bulk but of specific proteins). This second pathway is activated just when we have unfolded proteins.

These two pathways work not only when we have external signals; every time that we produce a protein, we can obtain a protein that does not work; what is changing is the amount of unfolded protein production. If the cell is able to manage the situation, the cell is more or less in normal conditions.

The problems comes when the systems are not properly working or when the number of the unfolded protein is very high, in this last case, they accumulate and forms aggregates, we can lose cell function that can lead to cell death.

We can have the UPR response both in the ER and in Mitochondria (we also have ribosomes) but they show different pathways that activate the system. In the ER (UPR_ER) we can distinguish three different ways, based on the action of three different molecules.

The first one is the IREα-UPR, if unfolded proteins accumulate in the ER, IREα dimerize and go under auto-phosphorylation, the system is activated and leads to the degradation of specific mRNA and miRNA (RIDD-> regulated IRE- dependent decay).

In the meantime, IREα promotes the unconventional splicing of XBP1 mRNA, and the XBP1 transcriptional factor is traduced better than that in normal conditions. The TF then goes to the nucleus and drives the expression of genes involved in

mitochondrial function through a process called mitochondrial protein synthesis. This process occurs in the mitochondrial matrix and involves the translation of mitochondrial DNA-encoded mRNAs into proteins. The proteins synthesized in the mitochondria are essential for various mitochondrial functions, including oxidative phosphorylation, the production of ATP, and the regulation of mitochondrial metabolism. In addition to protein synthesis, mitochondria also have their own quality control mechanisms to ensure the proper folding and degradation of mitochondrial proteins. This includes chaperone proteins that assist in protein folding, as well as proteases that degrade misfolded or damaged proteins. Overall, the proper functioning of protein trafficking, protein folding, and degradation pathways is crucial for maintaining cellular homeostasis and ensuring the health and function of organelles such as the endoplasmic reticulum and mitochondria.

functions (they have their own DNA), also in this case we have 3 different pathways (UPR-MT) that have the aim to down-regulate protein synthesis. The first way involves the ATF5 factor (accumulates in the cytosol), which is activated by the presence of unfolded proteins in the mitochondria. In the cytosol it interacts with other molecular partners such as CHOP and ATF4; they form a complex that can translocate to the nucleus to activate genes involved in proteases and chaperones production. [ATF4 is also involved in ER-UPR, when we have ER stress probably, we also have Mitochondria stress, so we can see reciprocal regulation/interaction] The second pathway is activated by the presence of unfolded proteins and ROS (overproduction or dysfunctional antioxidant system), SIRT3 is involved, this factor directly de-Acetylates mitochondrial proteins and indirectly lead the nuclear localization of FOXO3, which induces an antioxidant transcriptional program. The ER⍺-UPR is the last mechanism involved is

activated when unfolded proteins and ROS are in the mitochondrial intermembrane space; their presence activate AKT. AKT phosphorylates ER⍺(an estrogen receptor) which increase the proteasome(indirectly) and can also goes to the nucleus where it works as a transcriptional factor for specifics proteases

UPR-> are anabolic pathways

ERAD-> are catabolic pathways

The first ERAD pathway is the UBIQUITIN-PROTEASOME PATHWAY, in this way 3 different steps are required to reach the ubiquitination of proteins that must be degraded.

In the first step E1 binds Ub using an ATP molecule, and then the Ub is transferred to E2 (Ub conjugation enzyme).

In the 3rd step E3 carrying the protein binds it to the Ub. Then the protein (need 4 Ub residues) can go to the proteasome where it is degraded giving back amino acids.

The second ERAD pathway is the AUTOPHAGY, this process is not activated because of the activation of the proteasome, but autophagy is involved when the proteasome can't

properly work and/or when proteins can't be degraded. If all this process works correctly, the result is the restore of homeostasis. If something goes wrong, we start to accumulate unwanted proteins that can form undegradable aggregations, usually they cannot enter the proteasomes, but they can accumulate in autophagy structures (inhibits autophagy). If the aggregates can enter in some way the proteasome, it can be poisoned because the conditions (the aggregate) don't lead to degradation. We can have the same poisoning effects from the outside-> drugs. In both cases we will have unfolded protein aggregations.

We have several diseases related to ER [no need to remember all of them].

AMYLOIDOSIS

Is a condition occurring in several diseases associated with ER and mitochondrial stress. We can observe the presence in the EXTRACELLULAR compartment, of the deposition of AMORPHOUS INSOLUBLE FIBRILS (amyloid substance). That results in altered cell function-> the interaction between cells

When the tissue is impaired, the crosstalk with the environment is altered and also the architecture of the tissue (means mechanical change).

It was firstly analyzed with histological methods -> Congo red consent to see these structures (was an unknown substance), this color was usually used to color starch, for this reason they named it amyloid. Congo red staining shows green birefringence under polarized light. The protein implied must be Congo red positive that means that it has structure, fibers are unable to β-sheets branch and are undegradable.

We have several types of amyloid substance, which depends on the 90% for a specific amyloid protein, for the 15% of P component of SAP (this is common in most amyloid substances) and then the fibers are surrounded by glycosaminoglycans.

Pathogenesis -> Usually it depends on the production of abnormal amounts of proteins (due to unknown factors). Carcinogenic effects can be involved, the effect of this is that plasma cells are overproducing immunoglobulin light chains.

and they are accumulated extracellularly. Similar conditions can be found in chronic inflammation, there is an overproduction of acute phase proteins, one of them is the SAA (serum amyloid) that when overproduced is able to accumulate and aggregate becoming undegradable. The last condition is that we are producing the normal amount of proteins but there is one specific protein that has a mutation, that leads to a protein that cannot reach the correct folding, since the production is persistence, at a certain point the result is against the release of unfolded proteins aggregate. The typical structure of amyloid fiber is helicoidal and we can see where the color inserts, this conformation is producing the undegradable feature. Classification can be done by the localization in a specific organ (it could also be systemic) or according to the causes, primary amyloidosis (deriving from mutations, or situation that are directly intich in protein conformation) or secondary amyloidosis.

(deriving from chronic inflammations/diseases).

RED BLOOD CELL DISORDERS 18/10/2022

We can have different cell disorders, the major ones are the ANEMIA, consisting in a decreased erythrocyte number and the POLYCYTHEMIA, where the number of erythrocyte is increased, that can be a physiological condition (people living at high altitude have an increased erythrocyte number to compensate the low O2 concentration) but when it occur in the normal environment is pathological.

Every time we perform analysis, parameters are assessed, we can compare with the grid below and understand what's going on. They are markers of a healthy/diseased state.

Erythrocytes have the main function to be oxygen carriers, from the lung oxygen is carried in the peripheral tissues and then back to the lungs.

In HYPOXAEMIA conditions, the kidneys detect the low amount of oxygen in the blood and increased the production and secretion of ERYTHROPOIETIN, this molecule has different functions, firstly induce the production of

erythropoietin receptors (increase), and also stimulate the bone marrow to produce red blood cell precursor and induce their differentiation, resulting in an increase erythrocytes production. In physiological conditions, the increased erythropoietin levels is related to the activation of a specific transcriptional factor HIF1 (Hypoxia-inducible factor). Hypoxia stabilized the HIF1⍺. HIF1 phosphorylation lead to its stabilization, then HIF1⍺ and HIF1β form a dimers that can enter the nucleus where it forms a sort of transcriptional complex with other nuclear factor, activating a huge numbers of genes involved in different functions (increased blood flow, increase of ATP production etc.) The same pathway described (HIF) is activated when the low O2 concentration is due to erythrocytes number de