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Antitubulin Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Microtubules have an external diameter of 25 nm and a length varying from 250 nm to 25 µm. Their dynamic properties are based on their inherent structure and polarity, which is dependent upon the tubulin isoforms from which they derive. At present, six isoforms of tubulin are known within eukaryotic cells: alpha-(α), beta-(β), gamma-(γ), delta-(δ), epsilon-(ε), and zeta-(ζ) tubulins), with each isoform containing a number of subtypes. For example, the beta-(β) subtypes include TUBB, TUBB1, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4, TUBB4Q, TUBB6, and TUBB8. The alpha-(α) and beta-(β) isoforms form the structure of the polymeric form of tubulin which constitutes the microtubules, and γ-tubulin functions as a template for the correct assembly of microtubules. These isoforms, which each have a molecular weight of approximately 50 kDa, can be further modified by various post-translational modifications, including tyrosination and de-tyrosination, acetylation, polyglutamylation, polyglycylation, phosphorylation, and palmitoylation. Except for tubulin tyrosine ligase (the enzyme that adds a tyrosine to nonassembled α-tubulin), most of the modifying enzymes act preferentially on tubulin subunits that are already incorporated into microtubules. Post-translational modifications of tubulin subunits appear to mark sub-populations of microtubules, selectively affecting their functions. Although they are not directly involved in determining the dynamic properties of microtubules, modifications such as sequential tyrosination/de-tyrosination/acetylation correlate well with the half-life and spatial distribution of microtubules.
TANK-binding kinase 1 as a novel therapeutic target for viral diseases
Published in Expert Opinion on Therapeutic Targets, 2019
On the other hand, the formation of functional TBK1-containing complexes could be impaired by steric hindrance. A few of molecules block the interaction between TBK1 and its upstream adaptors and then prevent TBK1 activation, such as tubulin–tyrosine ligase-like protein 12 (TTLL12) [76], IFN-stimulated gene 56 (ISG56) [77], G patch domain-containing protein 3 (GPATCH3) [78], nucleotide binding domain and leucine-rich repeat-containing (NLR) family protein NLRC3 and NLRX1 [79,80], an alternatively spliced isoform of MITA termed MITA-related protein (MRP) [81], microtubule-interacting protein associated with TRAF3 (MIP-T3) [82] and suppressor of IKKε (SIKE) [83]. In addition, as a substrate of TBK1, SIKE also interacts with TBK1 with a higher affinity and thus suppresses TBK1-catalyzed IRF3 phosphorylation [84]. Protein arginine methyltransferases 6 (PRMT6) [85] and estrogen-related receptor α (ERRα) [86] inhibit the association of TBK1 and its substrate IRF3, resulting in turning off the transmission of the active TBK1 signal to IRF3.
RPGR gene therapy presents challenges in cloning the coding sequence
Published in Expert Opinion on Biological Therapy, 2020
Cristina Martinez-Fernandez De La Camara, Jasmina Cehajic-Kapetanovic, Robert E. MacLaren
Exon ORF15 is translated into a glutamic acid-glycine rich domain comprising glutamic acid and glycine residues as repeat-like sequence such as ‘EEEGEGEGE’. Due to the repetitive nature of the glutamic acid-glycine rich region and the fact that its length varies among species, it is challenging to decipher the function of this domain. This repetitive region contains 11 glutamate-rich consensus motifs. Indeed, RPGRORF15 has been found to be a glutamylation substrate and post-translational modification appears to be critical for its function [23,24]. The addition of negatively charged glutamates to this region is likely to affect the stabilization and the folding of the RPGRORF15 protein and its interaction with other proteins in the connecting cilia, particularly with regard to trafficking [25–27]. The connecting cilium represents a critical junction between the inner and the outer segments, allowing the bidirectional transport of opsins and other phototransduction proteins and contributing to the viability of the photoreceptors. Although RPGRORF15 function is not well understood, its interaction with other ciliary proteins to form protein complexes involved in cilia regulatory signaling pathways and the glutamylation of the ORF15 region in the C-terminus suggest that it plays an important role in microtubule-based transport to and from the basal bodies and within photoreceptor axonemes, contributing to the intracellular cargo movement between inner and outer segments (Figure 3) [23,28–30]. In humans, a lack of glutamylation caused by Tubulin Tyrosine Ligase Like 5 (TTLL5) enzyme deficiency causes an inherited retinal degeneration, similar to RPGR deficiency, hence indicating that the glutamylated protein is essential for normal RPGRORF15 function [23,31,32]. The acidic, repetitive glutamic acid-glycine rich domain is followed by a non-repetitive, basic domain in the carboxy-terminus. The basic domain is known to be evolutionarily conserved with a high sequence identity across species [33], suggesting that it too constitutes a functional domain.
Emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa
Published in Expert Opinion on Emerging Drugs, 2022
Cristina Martinez-Fernandez de la Camara, Jasmina Cehajic-Kapetanovic, Robert E. MacLaren
The activation of the proteins that constitute the regulatory network in the connecting cilia, and the large diversity of functions that these develop are mechanistically controlled by post-translational modifications. Specialized functions of microtubule-associated proteins have been related to glutamylation, glycylation, and tyrosination/detyrosination [78]. Glutamylation, the addition of glutamate side chains on glutamate residues catalyzed by Tubulin Tyrosine Ligase-like (TTLL) enzymes, contributes to the stabilization of microtubules and regulates the recruitment and activity of microtubule-interacting proteins [79]. RPGRORF15 is a glutamylation substrate [69,80]. Exon ORF15 is translated into a glutamic acid-glycine rich domain comprising G and E residues as repeat-like sequence such as ‘EEEGEGEGE'. This region can be considered as an intrinsically disordered region since it contains a high proportion of glutamic acid residues (60.1%) which could prevent a protein domain to mediate cooperative folding. The computation of various physical and chemical parameters of the ORF15 region determines that this unique sequence has an instability index of 87.52, which classifies this domain as unstable [81]. But what appears to be a disordered region with unstable structure, could be providing specialized functional advantages [82]. TTLL5 enzyme, expressed in rod and cone photoreceptors, interacts with the basic domain of RPGRORF15 and catalyzes the addition of glutamates to the 11 consensus motifs within the ORF15. The addition of negative charges to the ORF15 region might affect the stabilization of the RPGRORF15 protein, and the integrity of the RPGRORF15 containing protein complexes in the connecting cilia [69,83]. To date, there is no evidence that supports that RPGRORF15 can preserve fully its function with a reduced level of glutamylation and it has been suggested that a complete or partial lack of glutamylation may compromise RPGRORF15 function [80]. Indeed, loss-of-function variants in TTLL5, that lead to a lack of glutamylation, have been identified as a cause of recessive retinal dystrophy, similar to RPGR deficiency [49].