MUDr. Dana Maňasková


Vyhledávání na medicinman.cz
 

leky-latky/cftr-protein/mutace

Cystická fibróza

  • More than 2000 mutations have been identified
    • While 127 are confirmed as disease causing (Sosnay et al., 2013) [1]
  • Over 700 mutations that cause CF have been reported to the CF Genetic Analysis Consortium [17]
  • www.genet.sickkids.on.ca
  • Mutations that cause one or more problems
    • In the transcription, translation, protein processing, or channel activity of CFTR have been described [17]
  • Unfortunately, the vast majority of CF-causing mutations
    • Result in the absence or reduced expression of apical membrane CFTR protein. [17]

CFTR inhibition

  • Derangement of cellular proteostasis
  • CFTR malfunction increases the generation of reactive oxygen species (ROS)
    • Post-translational modifications of transglutaminase 2 (TGM2)
    • Versatile multifunctional enzyme that catalyzes several post-translational modifications of target proteins
      • TGM2 undergoes small ubiquitin like-modifier (SUMOylation)
        • Inhibits TGM2 ubiquitination leading to persistent high TGM2 protein levels a
        • TGM2 activation as the result of permissive elevated Ca2+ levels [25]
  • Activated TGM2 targets a plethora of substrates
    • Essential autophagy protein Beclin 1 (BECN1)
      • Essential for autophagosome formation [25]
    • Causes its dislodgement away from the endoplasmic reticulum,
      • Functional sequestration in intracellular aggregates [25]
  • Two major negative effects on cellular proteostasis
    • Functional sequestration of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3)
    • UV radiation resistance-associatedgene (UVRAG)
      • Two major components of the BECN1 complex
    • Negatively impacts on intracellular trafficking in CF epithelial cells
      • Reduces the availability of phosphatidyl-inositol-3-phosphate (PtdIns3P)
        • Perturbs endosomal fusion/maturation and trafficking [25]
  • BECN1 inactivation disables autophagy
    • Defective autophagosome formation
    • Accumulation of the autophagic substrate sequestosome 1 (SQSTM1) at endosomal level
    • Reduced availability of the small GTPase RAB5 and RAB7
      • Essential for endosomal maturation [25]
  • Ubiquitin-binding-protein SQSTM1 accumulates at the plasma membrane (PM)
    • Favors the disposal of several surface proteins, including
      • Epidermal growth factor receptor (EGFR)
      • CFTR itself [25]
  • Defective CFTR
    • Activation of the innate immune system at the mucosal surface
      • TGM2-mediated sequestration within histone-deacetylase 6 (HDAC6)+/vimentin+ intracellular aggresomes of the
        • Anti-inflammatory peroxisome proliferator-activated receptor-gamma
        • Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) inhibitor alpha (NFKBIA) [25]
  • The neutralization of NFKBIA
    • Nuclear translocation of the pro-inflammatory transcription factor of NF-?B
      • Increasing the transactivation of genes coding for pro-inflammatory cytokines
        • ROS generation [25]
  • Notably, CFTR, TGM2 and autophagy are engaged in a feed-forward loop [25]

High mobility group box-1 (HMGB1) - alarmin

  • Chromatin-linked, nonhistomic
  • Small protein with cytokine activity
  • Nuclear, cytosolic, and extracellular actions
  • Binds to
    • Chromosomal DNA
    • Toll-like receptor 3 (TLR3)
    • TLR4
    • Receptor for advanced glycation end products (RAGE)
      • That activates nuclear factor (NF)-kappaB
        • Upregulation of leukocyte adhesion molecules
        • Production of proinflammatory cytokines
        • Angiogenic factors
    • Promote inflammation
  • Well-recognized damage-associated molecular pattern (DAMP) protein
  • HMGB1 is increased both locally and in the circulation in conditions like
    • Obesity,
    • Cystic fibrosis,
    • Polycystic ovary,
    • Insulin resistance and diabetes
  • Produced by adipose tissue and the immune system
  • CFTR malfunction causes
    • Increase in HMGB1
    • Inflammation
    • Increased autophagy <5>(?) - různé od různých autorů
  • www.karger.com/Article/FullText/508291
  • Low-grade chronic inflammation
  • Could potentially be a biomarker of acute lung injury
    • Involved in COVID-19
  • HMGB1 expression is increased in thrombosis-related diseases
  • HMGB1, via RAGE, mediates sepsis-triggered amyloid-beta accumulation in diseases of the central nervous system
    • Associated with impaired cognitive function, e.g., neurodegenerative diseases
  • HMGB1 gene polymorphisms
    • Associated with hypertension in the Han Chinese population
      • Could be implicated in the outcome and course of COVID-19 in some individuals.
  • Mice that SARS-CoV downregulated ACE II protein, contributing to severe lung injury
  • ACE II overexpression reduce HMGB1
    • Besides reducing apoptosis in the myocardium postinfarction, in a rat model
  • Reduction in ACE II induced by the virus would in turn increase HMGB1
    • Thus contributing to the “cytokine storm” and the worst scenarios seen with COVID-19
  • Inflammasome mediates HMGB1 translocation from the nucleus to the cytoplasm
    • Subsequent release from the cell via type 1 interferon JAK/STAT1 activation
  • Pharmacological inhibition of JAK/STAT1 could be an approach for reducing circulating HMGB1
  • 2004, it was hypothesized that HMGB1 could play a possible pathogenic role in SARS-Cov1
  • CFTR malfunction in cystic fibrosis
    • Increases HMGB1 serum concentrations, along with inflammation
    • Further increases are observed at the onset of the specifically related diabetes
  • Need for assaying HMGB1 in the serum samples of COVID-19 patients
  • HMGB1 gene polymorphisms explain some of the variations

HMGB1 is recognized as a drug target

  • Salicylic acid (SA) derivatives
    • 3-aminoethyl SA
    • Amorfrutin B1
  • Methotrexate
  • Inflachromene
  • Glycyrrhizin
    • Have also been shown to lower HMGB1
    • 2003, in an in vitro model, a German group used glycyrrhizin to inhibit the replication of SARS-CoV1
      • As effective as ribavirin and mycophenolic acid
      • More effective than 6-azauridine and pyrazofurin
    • Confirmed in vitro using serum samples from patients
Nejméně toxické budou glycirhizin a kyselina salycilová...

Sweat test

  • Whereby conductivity of sweat is measured.
  • Those with defective CFTR have a high NaCl concentration due to the inability to resorb Cl- in sweat glands.
  • Normal sodium concentration in sweat is 30 mM
  • Subjects with >30 mM sodium are likely to have CF and those above 60 mM are positive for the disease (LeGrys et al. 2007)

Důsledky mutací

  • Isolated problems affecting the digestive or respiratory system
  • Some cases of idiopathic pancreatitis
    • Abdominal pain, nausea, vomiting, and fever
  • May be a risk factor, the cause of idiopathic pancreatitis is unknown.
  • Associated with rhinosinusitis - chronic inflammation of the tissues that line the sinuses
    • Sinus pain and pressure, headache, fever, and nasal congestion or drainage
  • Partially block the airways and interfere with breathing
  • Bronchiectasis
  • Allergic bronchopulmonary aspergillosis
    • Hypersensitivity to a certain type of fungal infection
  • medlineplus.gov/genetics/gene/cftr/#conditions

Hlen

  • Thick, sticky mucus accumulates making the airway prone to chronic infection and progressive inflammation

  • Impact of CFTR modulators
    • Chronic obstructive pulmonary disease, asthma, bronchitis,
    • Emphysema,
    • Pneumonia
    • Secretory diarrhea,
    • Polycystic kidney disease
    • Reproductive dysfunctions (congenital bilateral absence of the vas deferens) [17]
    • Testicular and sperm abnormalities [17]

Plíce

  • CFTR dysfunction leads to thickened mucus with airway surface liquid dehydration, airway obstruction, chronic infection, inflammation, and ultimately structural remodeling
  • insight.jci.org/articles/view/93029


The complement anaphylatoxins (C5a and C3a)

  • Important mediators of inflammation
  • C5a and C3a
    • Chemotactic for monocytes and granulocytes
    • Stimulate degranulation
    • Generation of reactive oxygen species (ROS) [24]
  • C5a
    • Particularly potent towards neutrophils
    • Critical for orchestrating their response towards pathogens [24]
  • C5a and C3a
    • Are elevated in the CF airway
    • Can be generated by non-complement proteases including NSPs
    • Correlate with markers of neutrophilic inflammation
      • Neutrophil count
      • CXCL8 in bronchoalveolar lavage (BAL) fluid from paediatric CF patients [24]
    • Generation of active C5a-like and C3a-like forms by NSPs
  • NSP-generated C5a-like fragments
    • Resistant to inactivation by carboxypeptidase B
      • An important regulator of C5a activity [24]
  • Interaction between C5a and soluble glycosaminoglycans (GAGs)
    • Abundant during chronic neutrophilic inflammation of the CF airway
    • GAG interaction influences C5a activity [24]

Role of macrophages in promoting CF disease progression

  • Elevated macrophage numbers are observed in the CF airway
  • Correlate with concentrations of CCL2 (macrophage chemoattractant) in CF BAL fluid (Bruscia and Bonfield 2016)
  • Stimulatory environment that could favour both M1 and M2 phenotypes (Bruscia and Bonfield 2016)
  • Levels of IL-4 and IL-13
    • Elevated in BAL fluid from CF patients
    • Promote polarisation of macrophages to the M2 phenotype (Hartl et al. 2006)
  • Pro-inflammatory cytokines
    • Indicative of M1 phenotype polarisation (IL-6, CXCL8 and TNFalpha) have also been reported to be elevated (Bonfield et al. 1995) [23]

Neutrofily

  • Including neutrophils, monocytes, and macrophages
  • Profoundly affected by the loss of CFTR function
  • Absence of CFTR appears to
    • Shift neutrophils and macrophages into a more activated state (Adib-Conquy et al., 2008; Zhang et al., 2018)
    • Augment both basal and stimulated cytokine release (Bonfield et al., 2012; Zhang et al., 2018; Lara-Reyna et al., 2020)
    • Hamper infection resolution (Bonfield et al., 2012)
    • Impair the production of agents that destroy phagocytosed pathogens (e.g., hypochlorous acid and reactive oxygen species; Zhou et al., 2013; Zhang et al., 2018).
  • www.frontiersin.org/articles/10.3389/fphys.2020.583862/full

Neutrofilní zánět

  • The CF lungs are susceptible to recurrent infection promoting chronic neutrophilic inflammation.
  • Neutrophils recruited to the CF lungs become dysfunctional
    • Are ineffective at clearing pathogens
    • Perpetuating inflammation
  • Neutrophil serine proteases (NSPs) released by neutrophils
    • Collaterally remodel the airway, reducing lung function and causing mortality. [23]

Complement anaphylatoxins (C5a and C3a)

  • C5a and C3a are elevated in the CF airway
  • Chemotactic for monocytes and granulocytes
  • Stimulate degranulation and the generation of ROS
  • Can be generated by non-complement proteases including NSPs
  • C5a and C3a correlate with markers of neutrophilic inflammation in bronchoalveolar lavage (BAL) fluid from paediatric CF patients
    • Neutrophil count
    • CXCL8 [23]

C5a

  • Potent towards neutrophils
  • Critical for orchestrating their response
  • Atypical C5a production by NSPs
    • Cannot be prevented by therapeutic complement inhibitors [23]
    • NSP-generated C5a-like fragments are resistant to inactivation by carboxypeptidase B [23]
  • Interaction between C5a and soluble glycosaminoglycans (GAGs)
    • Abundant during chronic neutrophilic inflammation of the CF airway
    • GAG interaction influences C5a activity [23]
  • CF airway environment modifies C5a function [23]

CFTR a okolní kanálky

  • Along mucosal membranes of CF patients, reduced Cl- secretion by CFTR
  • Induces Na+ resorption from the mucosal layer by ENaCs and
  • Subsequently Na+ secretion across the basolateral membrane (Donaldson and Boucher 2007)
  • Consequently, water is drawn from the mucosal layer through cell gap junctions
  • Causing dehydration of the mucosal surface (Donaldson and Boucher 2007).
  • Expression of Ca2+ activated Cl- channels (CaCC) on the apical surface of epithelial cells
    • Enables CF patients, with reduced CFTR activity, to maintain a level of hydration
    • That enables people without CFTR-mediated Cl- activity to survive (Donaldson and Boucher 2007)
    • Greater disease pathology in mice with genetically reduced CaCC expression (Clarke et al. 1994).
  • In mouse and human islets insulin and glucagon secretory defects in the absence of functional CFTR
  • Others report that CFTR expression is very low or absent in human pancreatic endocrine cells
    • Suggest that impairments in insulin secretion likely result from
      • Decreased islet mass
      • Intra-islet inflammation
      • Exocrine-derived inflammatory mediators
  • Repeatedly shown reductions in islet mass or beta cell area in animal models of CF
  • Beta cell area was reduced by as much as 50% in children less than 4 years old when compared with age-matched controls

  • CFTR activators increase water excretion into the extracellular space
  • May be beneficial in treating a number of conditions such as
    • Dry eye
    • Cholestasis,
    • Constipation
    • COPD
  • cftr-activators-and-inhibitors-300431506.html">www.prnewswire.com/news-releases/vanda-and-ucsf-announce-license-agreement-for-cftr-activators-and-inhibitors-300431506.html

Neutrofily a makrofágy

  • Mutations in CFTR also adversely impact innate and adaptive immunity
  • Dramatically reduce the ability of neutrophils and macrophages in the lungs to eliminate bacteria
  • Dramatically increase the levels of proinflammatory cytokines in the lungs, even in the absence of infection
  • Phenotypic variation in CF, even in cohorts with the same mutation in CFTR
    • Modifier genes, including
      • SLC26A9,
      • SLC9A3,
      • SLC6A14
      • TNF
      • TGFb1
      • Secondhand smoke
      • Pollutants
      • Clinical care
  • journals.physiology.org/doi/full/10.1152/ajplung.00225.2020
  • CF is highly heterogeneous
    • In patients with the same CFTR mutation genotype
      • Factors other than the CFTR gene itself contribute

Mutations in the CFTR gene

Interaction of immune and stromal cells in the CF

  • Pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-18, IL-6) by epithelial cells and macrophages
  • Stimulate the release of chemoattractants such as CXCL8 (IL-8) by other epithelial cells and macrophages but also T-cells.
  • Recruitment of neutrophil to the interstitial tissue
    • Release of matrix metalloproteases generates N-acetyl-PGP, recruiting more neutrophils
  • Necrotic neutrophils, not cleared by macrophages
    • Lyse and release DNA, protease and ROS
      • Promote airway obstruction and remodel the airway causing a loss of lung function. [23]

Inflammation can be initiated

  • By the recognition of:

Damage-associated molecular patterns (DAMPs)

  • These endogenous factors are released by necrotic or damaged cells following trauma (Rock et al. 2011)
  • Examples of DAMPs are
    • uric acid,
    • Heat shock protein 70
    • Fibrinogen domain A
    • Fragments of GAGs
    • CgG-rich deoxyribonucleic acid (DNA) regions (Rock et al. 2011) [23]

Pathogen-associated molecular patterns (PAMPs)

  • Exogenous factors released by pathogens
  • Stimulate the immune system and stromal cells
    • Bacterial components such as lipopolysaccharide (LPS)
    • Flagellin
    • Peptidoglycans
    • Unmethylated DNA (Tang et al. 2012) [23]

DAMPs and PAMPs

  • Recognised by pattern-recognition receptors (PRRs)
    • Expressed on the surface and within the cytosol of immune cells such as
      • T-cells, B-cells, NK-cells, dendritic cells, macrophages, and polymorphonuclear leukocytes (PMNs)(Tang et al. 2012) [23]

Toll-like receptors (TLRs)

  • TLR1, TLR2, TLR4 and TLR5
  • Recognise bacterial components
  • Up-regulate the expression and release of pro-inflammatory cytokines
    • Interleukin-1beta
    • Tumor necrosis factor-alpha (Tang et al. 2012) [23]

IL-1beta

  • Key pro-inflammatory mediator
  • Released through the activation of cytosolic complexes termed “inflammasomes” (Lamkanfi 2011)
    • Centered around NOD (nucleotide-binding oligomerisation domain) -like receptors (NLRs)
      • Can be primed and activated through stimulation of PRRs by DAMPs and PAMPs (Lamkanfi 2011) [23]
  • A major process of NLRP activation is
    • Expression of pro-IL-1beta
      • Activation by caspase 1
        • Stimulates the release of other pro-inflammatory mediators, amplifying inflammation (Lamkanfi 2011)
          • Recruiting leukocytes to the trauma site
          • Generation of ROS
          • Upregulating phagocytic receptors for phagocytosis of pathogens and debris [23]

Macrophages

  • Key cell type in the resolution of inflammation
  • Phagocytose cell debris and dead cells
  • Efferocytosis of neutrophils (Shapouri-Moghaddam et al. 2018)
  • Release IL-10 and transforming growth factor-beta
    • Simulate collagen expression in fibroblasts, establishing the extra cellular matrix (ECM) (Shapouri-Moghaddam et al. 2018)
    • IL-10 also suppresses and dampens inflammation
      • By inhibiting pro-inflammatory TLRs through upregulation of microRNA that target MyD88-dependent signaling (Mittal and Roche 2015) [23]

Zánět

  • Wt CFTR binds to and colocalizes with TRADD
  • TRADD is a key signaling intermediate connecting TNF? with activation of NFkappaB
  • DelF508 CFTR does not bind to TRADD
    • NF-kappaB activation is higher in CFBE expressing del.F508 CFTR than in cells expressing Wt CFTR
    • Effect is abolished when TRADD levels are knocked down
  • Binding of TNFalpha to its receptor
    • Stimulates the transcription factor NF-kappaB, P65
    • Downstream release of cytokines IL-6 and IL-8 into the airways of CF patients
  • TNFalpha receptor forms trimers
    • Cause the disassociation of the inhibitory protein SODD from the TNFalpha receptor
    • This allows the adaptor protein TRADD to bind to the death domain of the receptor
    • TRADD then forms a multi-protein complex
      • Ultimately causes phosphorylation of the inhibitory protein IkBalpha
        • Allowing NF-kappaB to translocate to the nucleus
          • Stimulates transcription of cytokines such as IL-8
  • www.karger.com/Article/FullText/453162

Deficit zinku plicního epitelu

  • Abnormalities in the ENaC and CFTR ion channels
    • Induced a reduction of zinc transporters
      • Causing a zinc deficiency in lung epithelial cells
  • This, in turn, activates MUC5AC, a mucin-producing protein.

O úroveň výše

Poslední aktualizace: 26. 2. 2021 14:05:26