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In order to maintain cellular homeostasis, a continual cycle of protein production and degradation is required. Protein level in cells is maintained through “proteostasis” which refers to integration of several cellular processes that monitors protein production, folding, trafficking, degradation, and clearance1. Eukaryotic cells have two main protein degradation mechanisms in place: the autophagy driven vacuolar (lysosomal) proteolysis and ubiquitin proteasome system (UPS)2. UPS is predominantly responsible for degradation of regulatory proteins whereas structural damaged proteins and organelles are degraded by autophagy in the lysosomes3 . An overlap between proteasomal degradation and autophagy has been observed which is seen to serve a compensatory function3. Research by Heinemeyer et al. on proteasome mutants successfully proved that UPS system plays a central role in degradation of redundant regulatory proteins, and serves as the main quality control mechanism present in cells4. Selective protein degradation by UPS provides a switch mechanism that can “switch off” or dismantle the existing regulatory network or ongoing cellular processes5. Rapid degradation of specific regulatory proteins is necessary for a wide range of dynamic cellular and developmental processes5. In addition, UPS serves as a key quality control to get rid of damaged or misfolded proteins in the cell. All proteins need to be folded in a specific three-dimensional conformation for them to be functional6. Often, mistakes occur in this well controlled mechanism leading to proteins being folded or refolded incorrectly6 2. This causes changes in the protein structure, resulting in defects in the protein function, protein aggregation and stresses of the cell6 2. UPS system controls degradation of misfolded proteins through Endoplasmic-reticulum-associated protein degradation (ERAD). Misfolded proteins are recognized, ubiquitinated in the endoplasmic reticulum and subsequently degraded by proteasome. Mutation in ERAD system leads to protein aggregation, which has been associated with several human diseases including Parkinson’s disease, Creutzfeldt-Jakob disease and cystic fibrosis7. This thesis paper will focus on UPS in context to its role in the immune system particularly in the NF-kB pathway and the therapeutic potential of targeting specific components of this pathway.
It is a protein tagging and destruction system present in all eukaryotes. It allows rapid and irreversible degradation of selective proteins, permitting the cell to precisely regulate and govern many processes such as cell cycle progression, apoptosis and immune response8.
UPS mediated selective protein degradation is achieved through a post translational modification-Ubiquitination, where ubiquitin (Ub) is covalently attached to substrate proteins via specific lysine residues 9. Ub is a small (8.5 kDa) evolutionary conserved 76 amino acid protein found in all eukaryotic cells9. Ub is linked to the specific target protein through a branched isopeptide bond10 between the α- carboxyl terminal glycine of ubiquitin and the Ԑ- amino group present on the lysine of the target substrate11. There are two ways in which Ub attaches to the substrate. A single Ub molecule is attached to the substrate protein to form monoubiquitination or multiple Ub-moieties are sequentially added to the Ub attached to substrate leading to formation of a polyubiquitin chain12 10. Ub has seven lysine residues all of which are capable of linking poly-Ub to form linkage-specific poly-Ub chains13. The K48-linked poly-Ub chains serve as a primary recognition signal for a downstream proteasome in UPS12. Poly-Ub chains linked through other lysine residues such as K63 serve different functions, one of which is signal transduction10 13. Monoubiquitination mediates many functions as well, such as receptor endocytosis, DNA repair and gene expression14.
Ubiquitination is carried out sequentially in three steps by a cascade of enzymes and molecules working together. It involves an integrative collaboration between E1 Ubiquitin-activating enzyme, E2 Ubiquitin-conjugating enzyme and E3 Ubiquitin-ligase enzyme15. Ubiquitination is initiated with Adenosine Triphosphate (ATP) dependent activation of Ub to a high energy intermediate on the ubiquitin-activating enzyme (E1)16 . Ub is attached to an internal E1 cysteine via an intermediate thiol ester generating E1-Ub complex. The high energy Ub-intermediate is then transferred to one of the several E2 enzymes via transthiolation to generate E2-Ub enzyme conjugate, which is connected via a thioester bond16. The substrate is then paired with one or more E3 Ub-ligases that facilitate Ub transfer to lysine side chain on the substrate protein (Refer to Figure 1)16. There are three types of eukaryotic E3 ligases: RING-type E3s, Homologous to E6aP C-Terminus (HECT)-type E3s and RING-in between-RING (RBR) E3s. Majority of E3s are RING-type ligases with a conserved RING/Ubox domain that binds to E2-Ub conjugate16. Diversity of Ub-protein ligase E3s are responsible for the high specificity and selectivity of the UPS system15 17.
(Dipankar et al, 2006) https://www.nature.com/libraries/catalog/
(Wang and Maldonado, 2006)
26S proteasome- It is a multi-subunit protease complex found in all eukaryotic cells. It is made up of 33 core proteins. The 2.0 MDa 26S proteasome is composed of the barrel-shaped 20S complex (700kD) capped by two 19S RP called PA700 in mammals18. The 19S RP are responsible for recognition of ubiquitinated proteins (with high fidelity), deubiquitylation (the ATP-dependent unfolding of polyubiquitylated substrates) and unfolding of Ub-bound substrate which is then transferred into the proteolytic central chamber for proteolysis15. The base contains six ATPase and caps the end of 20S proteasome core. It unfolds protein substrates and threads them into catalytic chamber of 20S particle in the presence of ATP19 20.
Substrate entry is a complex process, after the Ub-substrate binds 19S cap, the polyubiquitin chain is cleaved off the substrate and disassembled. The ATPases in the base of 20S particle unfold the substrate and threads them into catalytic chamber of 20S particle19 20. Linearization of protein is essential for its entry through 20S pore. Linearization and delivery of substrate requires significant energy and utilizes one-third of ATP as used by the ribosome during protein synthesis 19 20.
The barrel-shaped 20S proteasome complex is known as the core proteasome (CP) that is composed of two subunits in form of four stacked rings, a pair of identical inner α-rings and a pair of outer β-rings21. The β-rings contain many catalytic sites and houses the peptidase activity whereas, the function of the two outer α-rings is not well understood 21. In eukaryotes, two of the catalytic sites on β-rings are Chymotrypsin-Like (CTL) and two are trypsin-like (TL) and other two are caspase-like 22. Proteasome inhibitors can be used to target these sites to inhibit their proteolysis activity. All synthetic or natural proteasome inhibitors known today are known to act on the CTL sites15 22 . The linearized protein from RP is translocated to 20S central chamber where its six catalytic sites cleave the protein to form small peptides having lengths ranging from 3 to 25 residues 8. After complete digestion of the protein in small peptides they are released from the proteasome and are quickly digested to amino acids by cytosolic aminopeptidases which can then be utilized by the cell for other purposes. Sometimes, partial degradation of proteins by the proteasome can result in functional and biologically active fragment, such as the subunit of transcription factor NF-kB 8.
NF-kB is a major immune signaling pathway which uses UPS-dependent signaling23 and thereby, plays a vital role in activation of both innate and adaptive immunity. NF-kB controls immune cell development, B and T-cell differentiation, antigen and cytokine-induced intracellular pathways, inflammation and hematopoiesis23. Ubiquitination regulates the NF-kB signalling pathway in three major steps- Ub-proteasome led degradation of IkB proteins, processing of NF-kB precursors and IkB kinase (IKK) activation24 via phosphorylation, which will be discussed in more detail in the later sections.
NF-kB is an evolutionarily conserved transcription factor that plays an important role in regulation of many genes crucial for inflammation, lymphocyte activation, immune response, cell proliferation and apoptosis24. Initially, NF-kB was found to be constitutively present in only B cells and plasma cells but, it was later discovered to be present in all cell types25 and can be activated by appropriate stimuli. It also functions in virus proliferation in host cells as many viruses are known to exploit the NF-kB pathway. They control the pathway to activate NF-kB target genes and to ensure the survival of lymphoid cells where, the viruses grow and replicate. In addition, they also manipulate NF-kB pathway to degrade antiviral host proteins through regulation of host UPS26. There are several human diseases that manifest as a result of inappropriate regulation of various components of the NF-kB pathway including inflammatory and autoimmune diseases, cancer and neurodegenerative diseases. Therefore, a great deal of research has been conducted focusing on NF-kB and UPS as a therapeutic target for treatment of these diseases.
There are five members of the NF-kB family, NF-kB1 or p105/p50, NF-kB2 or p100/p52, RelA or p65, RelB and c-Rel 26(Refer to figure 2). All proteins of the NF-kB family share a highly conserved 300-amino acid Rel homology domain (RHD) located towards the N-terminus of the proteins27. The RHD domain containing a nuclear localization signal (NLS), allows the NF-kB proteins to dimerize, bind DNA in a sequence specific manner, and interact with inhibitory IkB proteins26 28. NF-kB family members attain different biological functions in the cell, which can be attributed to formation of NF-kB homo or heterodimers. They bind genes and activate transcription differentially29. NF-kB members are sequestered in the cytoplasm in a resting cell through their interaction with the NF-kB inhibitor protein I-kappa B (IkB)27. An immune response leads to degradation of the IkB proteins causing the NF-kB dimers to translocate into the nucleus where, they bind DNA corresponding to an enhancer and regulate gene transcription and expression27. Rel proteins such as p65 (RelA), Rel B and c-Rel all contain a C-terminal transactivation domain, which allows RelA and c-Rel to function as transcriptional activator30 and B-Rel to function as a repressor31.
p100 and p105 are precursors of the DNA binding subunits p52 and p50 respectively, that are post-translationally processed to become functional repressors. p50 or p52 also form heterodimers with Rel B proteins29. The C-terminus of the un-modified subunits (p105/p100) is known to have ankyrin repeats that share 65% similarity with sequence of IkB inhibitor29 32. This region undergoes ubiquitin-led degradation upon generation of p5229 and p5032 subunits. p50 plays varying roles in the NF-kB pathway. p50 homodimer (activator) can translocate into the nucleus, bind DNA and activate gene transcription. p50 heterodimerization to p65 produces the p50-p65-IkB ternary complex. This predominant heterodimer prevents p50 translocation into the nucleus thereby, inhibiting gene transcription32.
Nuclear translocation of the NF-kB dimers and DNA binding results in transcription of its target genes such as chemokines, cytokines, adhesion molecules, inflammatory mediator producing enzymes and inhibitors of apoptosis29. These molecules are important components of the innate immune response to invading microorganisms and are required for migration of inflammatory and phagocytic cells to tissues where NF-kB has been activated in response to infection or injury. These proteins lead to recruitment of inflammatory and phagocytic cells to the site of infection and activate them once they have reached their target. The activated phagocytic cells kill and clear the infection or damaged cells33. They also play a major role in other aspects of immune system such as antigen presentation and activation of adaptive immune system. This is achieved by presentation of degraded short polypeptides on cell surface by MHC class I molecules. This allows cells presenting peptides derived from foreign proteins including viral proteins to be recognized by cytotoxic T-lymphocytes and be lysed 28.
In resting cells, the dimeric NF-kB proteins are generally localized in the cytoplasm through their association with NF-kB inhibitor protein IkB. IkB protein family is composed of IkBα, IkBβ, IkBγ, IkBԐ, IkBsomes, Bc1-334. The inhibitory proteins all have 6-7 ankyrin repeat domains which recognize and bind to the RHD of the NF-kB Rel proteins26. IkB inhibits their DNA binding ability by masking their NLS, causing retention of NF-kB (transcription activator) in the cytoplasm35. NF-kB dimers exhibit varying degrees of affinity to the kB DNA sites that have the sequence motif of 5′-GGGRNNYYCC-3′ (where purine A or G is represented by R; purine A, C or T is represented by H and pyrimidine C or T is represented by Y)36 26. IkB is also phosphorylated in response to external stimuli by the Ik kinase (IKK) complex which is a large molecular weight kinase37. This signal induced IkB phosphorylation leads to IkB degradation and activation of the NF-kB canonical pathway38. The IKK complex is composed of two subunits, IKKα (IKK1) and IKKβ (IKK2), and a regulatory subunit called NF-Kb Essential Modulator (NEMO) or IKKγ39. NF-kB activation requires IkB phosphorylation. Cytoplasmic IKK catalyzes the phosphorylation of IkBα at Ser 32 and Ser 36 and/or phosphorylation of IkBβ at Ser 19 and Ser 2328. Phosphorylation of IkB stimulates polyubiquitination of IkB protein, leading to subsequent degradation by the 26S proteasome, which allows release and translocation of NF-kB into nucleus to initiate gene transcription37. Researchers have identified at least two different pathways for NF-kB activation: The canonical pathway and the alternate pathway. The two pathways are activated by varying stimuli and utilize different subunits of the IKK complex28. For example, in canonical pathway IKK complex only requires IKKβ/IKK2 and IKKγ but does not require IKK1 whereas, activation of alternate pathway requires IKK1 but is independent of IKK2 and IKKγ33.
(Shih-VF et al 2011)
The Canonical pathway:
The “Canonical pathway” or classical pathway is activated via multiple stimuli: 1)proinflammatory cytokines: Interleukin-1 (IL-1) and Tumor Necrosis Factor- α (TNF- α)33. IL-1 and TNF- α are released upon infection, injury or during certain inflammatory diseases such as Rheumatoid Arthritis (RA), Immune Bowel Disease, Asthma and Chronic Obstructive Pulmonary Disease33. 2) Pathogen-associated molecular patterns
There are many different types of intra and extra cellular receptors that these molecules stimulate. For example, Toll-like receptors (TLR), CD40 ligand (CD154), receptor activator of NF-kB ligand (RANKL), major histocompatibility (MHC) molecules and peptide, and antigen–antibody cross-linking the B cell receptor (BCR)40. TLRs recognize microbial molecular patterns and encourages binding33.
NF-kB Canonical mechanism-
As we know that in a resting cell, NF-kB dimers p50-RelA are sequestered in the cytoplasm by their association with IkB proteins. External stimuli triggers activation of IKK complex leading to the phosphorylation of serine/threonine kinase -the IkB members (IkBα, IkBβ, IkBԐ and p105) on the consensus motif DSGFxS. Phosphorylation of IκBα on the N-terminal serines 32 and 36 causes recruitment of SCFβTRCP E3 Ub-ligase, the substrate recognition subunit of an E3 ligase named SCFβ-TrCP41. This leads to ubiquitination of a specific lysine. This is followed by full degradation of ubiquitinated IkBs by proteasome leading to nuclear translocation of the NF-kB complexes thereby, activating NF-kB mediated gene transcription42. The Canonical pathway is mediated by IkBβ and NEMO-dependent IKK (IKKγ)35 subunits of the IKK complex. Studies conducted on NF-kB RelA/p65 subunit deficient mice resulted in embryonic lethality in the liver caused by massive apoptosis43. This showed that RelA/p65 controlled essential transcription in NF-kB pathway and disruption in NF-kB transcription caused functional defects in cells. A similar experiment where gene encoding IKK2 was inactivated to produce IKK2 mutant mice also displayed embryonic lethality44. Obtaning similar results from both IKK2 and RelA knockouts showing that both of these molecules are essential components of the NEMO-IKK2-RelA pathway which is characterized as the canonical pathway.
Studies have also shown the role of NF-kB in apoptosis regulation where RelA was shown to be crucial for TNF-mediated apoptosis in macrophages, hepatocytes and T-cells 45 42. In recent years, IKKα has been shown to use it as a compensatory mechanism in absence of the IKK2. However, recent work suggests that IKK1 plays a role in the canonical pathway as well, and may compensate for the loss of IKK2 within the NEMO-dependent kinase.
Figure 4: Canonical NF-kB pathway- This figure illustrates the canonical pathway for NF-kB activation. The canonical pathway is activated by TLRs and TNF-α and IL-1 (proinflammatory cytokines). Stimulation of receptor causes phosphorylation of IkB complex comprising of IkBα, IkBβ and NEMO (IkBγ). This causes subsequent phosphorylation of IkB leading to its ubiquitination and proteasome led degradation. Degradation of IkB frees the bound RelA-p50 dimer which translocates into the nucleus. Activated RelA regulates expression of proinflammatory and cell survival genes.
Alternate NF-kB pathway-
Among the inducers of the classical NF-kB pathway, few of them are able to trigger an additional pathway through the activation of the NF-kB-inducing kinase (NIK) and IKKα. The “alternative pathway” is activated by TNF family of cytokines such as lymphotoxinβ, CD40 ligand, B-cell activating factor, and receptor activator ligand except TNF-α and leads to the activation of RelB/p52 complexes42.
The recently discovered “alternate pathway” could activate RelA/p65 independent of NEMO-dependent kinase and requires NF-kB inducing kinase (NIK) for activation46. This pathway drives the post-translational processing of p100 to produce mature p52. Strikingly, IKKγ is not absolutely required for the activation of the alternative NF-kB pathway, which suggests the existence of an alternative IKKα-containing complex (two IKK1/IKKα). Although, p100 is also an SCFb-TrCP E3 ligase substrate, ubiquitinated p100 is not completely degraded by the proteasome but rather cleaved to generate active DNA-binding p52 product. This process is generally slower than the activation of the classical pathway and leads to a delayed activation of nuclear p52-containing complexes, such as p52/RelB. The mechanisms of generation of p52 are, either constitutive or inducible, either co-translational or post-translational, and take place in different cellular compartments. The alternative NF-kB pathway is characterized by the inducible phosphorylation of p100 by IKKα, leading to activation of RelB/p52 heterodimers. The upstream kinase that activates IKKα in this pathway has been identified as an NIK (NF-kB inducing kinase)42. Genetic studies in mice have showed the important role for this pathway in lymphoid organogenesis and B-lymphocyte function. Its role in inflammation is still unclear.
Figure 4: The Classical and Alternate NF-kB pathway- a) Canonical pathway is activated via TLRs, proinflammatory cytokines, viruses present on the cell surface. TNF-α and IL-1b activates the receptors. This leads to a cascade of signaling through phosphorylation of IKK complex (IkBα, IkBβ, IkBγ) and IkB inhibitor leading to its ubiquitination and degradation. Activated p50-p65 complex translocates into nucleus and coordinates expression of many inflammatory and innate immunity genes such as cytokines, chemokines, enzymes, and adhesion molecules. This leads to formation of amplifying feed forward loop.
b) The Alternative pathway is activated via cytokines such as LTβR, BAFF and CD40L by NIK. This causes phosphorylation of IKKα homodimers which in turn causes phosphorylation of Relb/p52 complex. The freed Relb/p52 translocates into the nucleus and coordinates expression of chemokines, cytokines and is required for lymph-organogenesis and B-cell activation.
As established above, we now know that aberrant NF-kB pathway plays a major role in origin of number of autoimmune and inflammatory diseases. Many of these diseases are caused by defects in the UPS system that plays a central role in NF-kB activation. As a result, a lot of research has been conducted to test Proteasome Inhibitor efficacy in inhibiting the aberrant NF-kB pathways by preventing proteasome degradation which causes the subsequent NF-kB activation. Proteasome inhibitor development started more than 40 years ago47. They have been a subject of interest for their therapeutic potential to treat cancers, immune and inflammatory diseases associated with defects in the NF-kB pathway. Additionally, proteasome inhibitors have served as major game changers by being used as an experimental tool to help researchers better understand cell regulation, immune surveillance and disease mechanisms47.
Bortezomib (BTZ) is a boronic peptide has been proven to be one of the most successful drugs that has made a major difference in the treatment of multiple myeloma (MM)- a cancer of plasma cells which are responsible for production of antibodies. Myeloma cells respond to proteasome inhibition because they have increased activity of the NF-kB pathway and one observes an accumulation of defective immunoglobulins which, would generally be degraded in normal cells by UPS as part of cell’s quality control mechanisms thereby, keeping cells free of defective immunoglobulins. Moreover, NF-kB transcription factor promotes expression of cytokines and growth factors and inhibits apoptosis thereby, associated with tumor pathogenesis. NF-kB remains in an inactive state in cytoplasm through its association with IkB inhibitors. IkB inhibitors serve as a key molecular targets for regulating NF-kB activation48. Since, Degradation of IkB by UPS leads to inhibition of apoptosis, usage of proteasome inhibitor that retain the IkB-NF-kB complex in the nucleus leads to excessive accumulation of misfolded proteins in the cell cytoplasm augmenting ER stress which in turn leads to apoptosis of cancer cells48 49.
BTZ (PS-341) has successfully become one of the most used FDA approved proteasome inhibitor for treatment of MM today. BTZ or Velcade as known commercially selectively blocks the 26S chymotrypsin activity of the proteasome49 by forming reversible and less specific tetrahedral adducts with Threonine1 site of catalytic β5 subunits50 of proteasome. BTZ causes accumulation of tumor suppressor like p53 and p27(KIPI) 51 and accumulation of BAX which is a pro-apoptotic protein52. However, it is limited by the dosage amount due to BTZ-induced side effects 53. It also binds to serine proteases that could lead to neurotoxicity53. Patients have also seen primary and secondary resistance to BTZ which has led to development of a second generation of proteasome inhibitors that are more effective and less toxic such as Carfilzomib (CFZ) 53 or Kyprolis. It is a tetrapeptide epoxyketone which forms stable and irreversible connections and inhibits the chymotrypsin site and caspase-like sites on the proteasome53 blocking access of substrate proteins to catalytic sites. They overcome serine degradation as non-proteasomal serine lacks the α-amino site that is responsible for binding with CFZ, decreasing the risk of neurotoxicity54 55 and providing a higher specificity, potency to CFZ compared to BTZ. Both, of these drugs require systematic administration, therefore, it would be better to discover better orally administered alternatives or improving their bioavailability. Another PI delanzomib has better pharmacokinetic properties compared to BTZ which can also be administered orally56. Velcade has shown great efficacy in treatment of MM and other cancers57 by targeting UPS using proteasome inhibitors, but proteasome inhibitors are much more non-specific. Use of non-specific proteasome inhibitors has been shown to affect the rate of normal protein turnover. Moreover, one can also observe severe side effects including neuropathy, thrombocytopenia, severe hepatitis, pulmonary fibrosis and respiratory failure58. A decrease in the rate of development of BTZ’s and other proteasome inhibitors to treat inflammatory and autoimmune diseases can be observed. with increase in the rate of BTZ resistance and decrease in the rate of any new development of proteasome inhibitors, the discovery and development of new drugs takes precedence.
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