Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has ...been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of ∼1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed.
Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research ...area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely nonlysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.
The ubiquitin–proteasome system (UPS) is involved in selective targeting of innumerable cellular proteins through a complex pathway that plays important roles in a broad array of processes. An ...important step in the proteolytic cascade is specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed that the first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to an ε-NH
2 group of an internal Lys residue. Recent findings indicate that, for several proteins, the first ubiquitin moiety is fused linearly to the α-NH
2 group of the N-terminal residue. An important biological question relates to the evolutionary requirement for an alternative mode of ubiquitination.
Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several ...proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine--a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.
Until the early 1980s, protein degradation was a neglected research area, and scientists were mostly busy deciphering the genetic code and its translation to the proteome. Destruction of cellular ...proteins was thought to be a scavenger, non-specific and dead-end process. Although it was known that proteins do turn over, the large extent and high specificity of the process, whereby distinct proteins have half-lives that range from a few minutes to several days, was not appreciated. The discovery of the lysosome by Christian de Duve did not change this view significantly, as it was clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate-specific. The discovery of the complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that degradation of cellular proteins via the ubiquitin system is a highly complex, temporally controlled and tightly regulated process that plays major roles in a variety of basic pathways and processes during cell life and death, and in health and disease. The system is involved in targeting many cellular proteins, among them cell cycle regulators, growth- and differentiation-controlling factors, transcriptional activators, cell-surface receptors and ion channels, endoplasmic reticulum proteins, antigenic proteins destined for presentation on class I MHC molecules, and abnormal/misfolded proteins. Consequently, it is involved in regulating many basic cellular processes, such as cell cycle and division, growth and differentiation, signal transduction and transcription, modulation of the secretory and endocytic pathways, the immune and inflammatory responses, and quality control. With the multitude of substrates targeted and the numerous processes involved, it is not surprising that aberrations in the pathway have been implicated in the pathogenesis of many diseases, with certain malignancies and neurodegenerative disorders being ranked among them.
The HECT-type E3 ubiquitin ligase (E3) Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease, including lung and stomach inflammation and hyperplasia ...of lymphoid and hematopoietic cells. The involvement of Itch in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review aims to bring together a growing body of work exploring Itch-regulated biological processes, and to highlight recent discoveries on the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Our contribution is also an endeavor to correlate Itch substrate specificity with the pathological defects manifested by the mutant Itchy mice.
Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded ...by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.
A path toward understanding neurodegeneration Kosik, K. S.; Sejnowski, T. J.; Raichle, M. E. ...
Science (American Association for the Advancement of Science),
08/2016, Letnik:
353, Številka:
6302
Journal Article
Recenzirano
Odprti dostop
The specter of neurodegenerative disease, particularly Alzheimer's disease, haunts the developed world and exacts a poorly documented toll on underdeveloped countries. With so little progress made ...toward finding a cure-or, better, a prevention-it is time to rethink the path to progress. This requires a change in perspective on the type of research that will make a difference. The lesson learned from cancer research is that a new commitment means rethinking the fundamental approach to the disease. Cancer research moved from taking potshots with, usually, cytotoxic drugs to a bottom-up, mechanism-based approach in which newly acquired genetic knowledge played the largest role. Today, that effort has produced a platform of knowledge from which academia and industry are drawing. For neurodegenerative disease, the genetic approach remains valid but the problem must concurrently be approached from a complementary, robust cell biological perspective, focusing on the cellular cascade of events that lead to neuronal cell death.
How the genetic code is translated into proteins was a key focus of biological research before the 1980s, but how these proteins are degraded remained a neglected area. With the discovery of the ...lysosome, it was suggested that cellular proteins are degraded in this organelle. However, several independent lines of experimental evidence strongly indicated that non-lysosomal pathways have an important role in intracellular proteolysis, although their identity and mechanisms of action remained obscure. The discovery of the ubiquitin-proteasome system resolved this enigma.
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