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The Ubiquitin-Proteosome System: A Critical Regulator in Pulmonary Fibrosis

Introduction

Idiopathic pulmonary fibrosis (IPF)  is a chronic and irreversible disease of the lungs that presents in patients as ongoing cough and dyspnea (uncomfortable breathing) for a prolonged period without other underlying lung disease, and has been estimated to affect over 3 million people worldwide1,2. This progressive disease is characterized by accumulating scarring of the lungs that diminishes quality of life, and has a 3-5 year survival time from diagnosis when untreated3.  Currently, nintedanib and pirfenidone (anti-fibrotic drugs) are the only approved treatments for IPF, and while they may extend survival by a few years with early intervention, they will not reverse the scarring and damage to the lungs already present4.  The molecular mechanisms that regulate IPF are not well defined but are presumed to be triggered by repetitive injury to the lungs and correlate with the most considerable risk factor for IPF which is aging. At the cellular level, mechanisms that affect cell aging and senescence include processes like telomere attrition and DNA damage, protein recycling and degradation pathways, and mitochondrial dysfunction5,6.  Interestingly, several studies have linked the ubiquitin-proteasome system to IPF, and this newsletter will delve into why ubiquitination has emerged as a critical regulator of this disease.

The Ubiquitin-Proteasome System in IPF

Ubiquitination is a well-studied post-translational modification (PTM) that regulates numerous cellular events and functions through mechanisms like mono-ubiquitination and its well-established role of poly-ubiquitinating proteins to target them for degradation via the proteasome.  Specifically, the ubiquitin-proteasome system (UPS) targets proteins through the classical E1, E2, and E3 enzyme cascade to attach ubiquitin (Ub) to key lysine residues on target proteins and mark them for degradation. E3 ligases, which number in the hundreds, are critical for target protein selection.  Conversely, deubiquitinases (DUBs) can enzymatically remove ubiquitin from target proteins and lower the overall ubiquitination state in cells.  Both Ub ligases and DUBs have been investigated in the setting of IPF and have been shown to affect disease progression7,8.  For example, E3 ligases such as BARD1, HRD1, and MDM2 were shown to have misregulated expression levels in IPF and had a variety of effects on cells including dysregulated proliferation and apoptosis through disruption of target protein signaling (see Figure 1)(reviewed in 7, 8).  In contrast, the DUB USP13, which regulates PTEN ubiquitination, was decreased in patients with IPF and alterations in USP13 and PTEN levels enhanced Fibrotic phenotypes9.

November_Newsletter_Figure-01

Figure Legend: Both TGF-β-dependent and independent pathways contribute to the pathogenesis of IPF. Ubiquitin E3 ligases, such as FIEL1, arkadia, TIF1γ, and STUB1 regulate TGF-β/TβR/SMAD pathways, while ubiquitin E3 ligases, MDM2, BARD1, and HRD1 regulate SMAD-independent pathways.

TGF-β1 and Poly-Ubiquitination in Fibrosis

TGF-β-signaling has been identified as a central player in IPF progression and functions through mechanisms including stimulating extracellular matrix production, epithelial-to-mesenchymal transition of alveolar epithelial cells, and accumulation of myofibroblasts10. Several Ub ligases like Arkadia, STUB1, and FIEL1 were shown to alter IPF progression via TGF-β1-dependent mechanisms(reviewed in 7, 8).  Lear et al, showed that dual phosphorylation of FIEL1 and PIAS4 resulted in FIEL-regulated ubiquitination of PIAS4 to control TGF-β levels11. Of importance, FIEL1 was shown to be elevated in the lungs of IPF patients, and knockdown of FIEL1 dampened fibrosis progression11.  Similarly, Arkadia also affected IPF through the regulation of TGF-β; however, it targets the TGF-β inhibitory protein SMAD7 for degradation in fibrosis12. Nedd4, another E3 ligase, was shown to be important in IPF.  Nedd4L was specifically knocked out in lung epithelial cells using a conditional knockout system, and this depletion led to lung disease with features of IPF including progressive fibrosis13. In a recent study, Nedd4L was also shown to be depleted in the lungs of IPF patients and in TGF-β1-treated lung fibroblasts; this depletion was rescued by inhibition of the transcription factor E2F14.  A feedback loop between Nedd4L and TGF-β1 exists, as the E3 ligase targets the TGF-β receptor II (TβRII) for degradation. Nedd4 has also been shown to target other proteins via ubiquitination such as YY1 to control IPF progression15, further implicating it as a critical player in pulmonary fibrosis.  This feedback loop between TGF-β1 and Nedd4 isn’t isolated, as another recent study determined that overexpression of the thioredoxin-interacting protein (TXNIP) attenuated TGF-β1 signaling in the setting of pulmonary fibrosis while also showing that TGF-β1 induced TXNIP proteasomal degradation via a USP13 DUB dependent mechanism16.  In a complimentary study to the evidence showing that Nedd4 can target TβRII for degradation, another study identified USP11 as the DUB that deubiquitinates TβRII to stabilize TGF-β1 signaling, and utilization of the USP11 inhibitor, mitoxantrone, diminished TGF-β1 signaling and suppressed fibrosis progression in cell models17.  In one final example, ubiquitin carboxyl-terminal hydrolase-L5 (UCHL5) DUB deubiquitinated Smad2 and Smad 3 to stabilize these TGF-β1 signaling proteins which promoted progression of fibrosis in IPF models.

Lung Fibrosis and Pulmonary Hypertension

Pulmonary hypertension (PH) is a disease characterized by an increase in pulmonary artery pressure, adverse vascular remodeling, stiffening of the pulmonary arteries, hypertrophy, and right ventricle failure18.  There are 5 classifications of PH with varying degrees of occurrence 1. Pulmonary arterial hypertension (PAH) (rare occurrence), 2. PH associated with left-sided heart disease (very common occurrence), 3. PH associated with lung disease (common occurrence).  PH associated with pulmonary artery obstructions (rare occurrence), and 5. PH with unclear or multifactorial mechanisms (rare occurrence), and when these groups are combined PH is estimated to affect 1% of the population18.  With regards to IPF, it has been shown that in patients with both IPF and PH, the outcomes for these patients with both diseases is always worse19.  Identifying common dysregulated signaling mechanisms may have particular benefits for these patients especially as PH with associated lung disease is a common occurrence as noted above. To that end, a recent study by Fan et al. identified the transcription factor Twist1 as an important regulatory protein for controlling PH20.  Furthermore, in models of PH depletion of Twist1 attenuated PH progression20.  The study found that Twist1 regulated GATA6 protein levels and BMPR2 via an MDM2-dependent ubiquitination mechanism.  Beyond the ubiquitination link, Twist1 has previously been found to be highly upregulated in IPF21.There is also growing evidence that UPS is involved in PH, and a recent preprint highlights some of the same ligases that regulate IPF may also affect PH.  One example is a study by Shen et al. that showed that MDM2 was increased in patients with PAH22.  They determined that MDM2 ubiquitinated angiotensin-converting enzyme 2 (ACE2) to alter PH progression.  A very recent study investigated the role of DUBs in the setting of PAH and found that the ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) DUB protein deficiency was sufficient to attenuate PAH23.

Summary and Future Perspectives

The studies highlighted here cumulatively support the importance of the UPS in the progression of IPF.  It is interesting that multiple Ub ligases and DUBs have been identified to independently regulate both TGF-β-dependent and independent mechanisms in the setting of fibrosis, and further investigation is warranted to identify if manipulating a single ligase or DUB is sufficient to suppress IPF progression.  Furthermore, it will be interesting to determine if altering the UPS through therapeutic intervention will reverse the effects of fibrosis on the lung.  Also of interest is the potential for UPS signaling pathways that regulate both IPF and some forms of PH.  As these diseases can occur simultaneously and result in worse outcomes, it will be of great benefit if targeting the UPS could provide simultaneous therapeutic benefits.

References

  1. Guo, H., et al., Progress in understanding and treating idiopathic pulmonary fibrosis: recent insights and emerging therapies. Front Pharmacol, 2023. 14: p. 1205948.
  2. Martinez, F.J., et al., Idiopathic pulmonary fibrosis. Nat Rev Dis Primers, 2017. 3: p. 17074.
  3. Alsomali, H., et al., Early Diagnosis and Treatment of Idiopathic Pulmonary Fibrosis: A Narrative Review. Pulm Ther, 2023. 9(2): p. 177-193.
  4. Zhao, C., et al., Drug therapies for treatment of idiopathic pulmonary fibrosis: a systematic review, Bayesian network meta-analysis, and cost-effectiveness analysis. EClinicalMedicine, 2023. 61: p. 102071.
  5. Hamazaki, J. and S. Murata, Relationships between protein degradation, cellular senescence, and organismal aging. J Biochem, 2024. 175(5): p. 473-480.
  6. Di Micco, R., et al., Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat Rev Mol Cell Biol, 2021. 22(2): p. 75-95.
  7. Li, S., et al., Ubiquitination and deubiquitination emerge as players in idiopathic pulmonary fibrosis pathogenesis and treatment. JCI Insight, 2018. 3(10).
  8. Inui, N., S. Sakai, and M. Kitagawa, Molecular Pathogenesis of Pulmonary Fibrosis, with Focus on Pathways Related to TGF-beta and the Ubiquitin-Proteasome Pathway. Int J Mol Sci, 2021. 22(11).
  9. Geng, J., et al., Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis. Respir Res, 2015. 16: p. 124.
  10. Yue, X., B. Shan, and J.A. Lasky, TGF-beta: Titan of Lung Fibrogenesis. Curr Enzym Inhib, 2010. 6(2).
  11. Lear, T., et al., Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4. J Exp Med, 2016. 213(6): p. 1029-46.
  12. Koinuma, D., et al., Arkadia amplifies TGF-beta superfamily signalling through degradation of Smad7. EMBO J, 2003. 22(24): p. 6458-70.
  13. Duerr, J., et al., Conditional deletion of Nedd4-2 in lung epithelial cells causes progressive pulmonary fibrosis in adult mice. Nat Commun, 2020. 11(1): p. 2012.
  14. Li, S., et al., Nedd4L suppression in lung fibroblasts facilitates pathogenesis of lung fibrosis. Transl Res, 2023. 253: p. 1-7.
  15. Chen, L., et al., Involvement of E3 ubiquitin ligase NEDD4-mediated YY1 ubiquitination in alleviating idiopathic pulmonary fibrosis. Int J Biol Macromol, 2024. 269(Pt 2): p. 131976.
  16. Taleb, S.J., et al., Molecular Regulation of Transforming Growth Factor-beta1-induced Thioredoxin-interacting Protein Ubiquitination and Proteasomal Degradation in Lung Fibroblasts: Implication in Pulmonary Fibrosis. J Respir Biol Transl Med, 2024. 1(1).
  17. Jacko, A.M., et al., De-ubiquitinating enzyme, USP11, promotes transforming growth factor beta-1 signaling through stabilization of transforming growth factor beta receptor II. Cell Death Dis, 2016. 7(11): p. e2474.
  18. Mocumbi, A., et al., Pulmonary hypertension. Nat Rev Dis Primers, 2024. 10(1): p. 1.
  19. Rajagopal, K., et al., Idiopathic pulmonary fibrosis and pulmonary hypertension: Heracles meets the Hydra. Br J Pharmacol, 2021. 178(1): p. 172-186.
  20. Fan, Y., et al., TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2. Am J Respir Crit Care Med, 2020. 202(9): p. 1283-1296.
  21. Bridges, R.S., et al., Gene expression profiling of pulmonary fibrosis identifies Twist1 as an antiapoptotic molecular "rectifier" of growth factor signaling. Am J Pathol, 2009. 175(6): p. 2351-61.
  22. Shen, H., et al., MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension. Circulation, 2020. 142(12): p. 1190-1204.
  23. Tang, H., et al., Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension. Circulation, 2024. 150(4): p. 302-316.
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