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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 642-645, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0200WS

NHLBI and ORD Workshop

Protein Processing and Degradation in Pulmonary Health and Disease

Pamela L. Zeitlin, Dorothy Berlin Gail and Susan Banks-Schlegel

Eudowood Division of Pediatric Respiratory Medicine, Department of Pediatrics, John Hopkins School of Medicine, Baltimore; and Division of Lung Diseases, National Heart, Lung, and Blood Institute, Bethesda, Maryland

Address correspondence to: Susan Banks-Schlegel, Ph.D., Division of Lung Diseases, National Heart, Lung, and Blood Institute, Two Rockledge Center, Suite 10018, 6701 Rockledge Drive, MSC 7952, Bethesda, MD 20892-7952. E-mail: Schleges{at}nih.gov

Abbreviations: {alpha}1-antitrypsin, {alpha}1-AT • cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • endoplasmic reticulum, ER


   Introduction
Top
 Introduction
Protein Degradation and Lung...
 Future Directions
 References
 
Cystic fibrosis (CF), {alpha}1-antitrypsin ({alpha}1-AT) deficiency, and some forms of interstitial lung disease involve misfolding and abnormal processing of proteins. Proteins are tagged for degradation in the endoplasmic reticulum (ER) by a multistep pathway that targets the proteins for destruction (Figure 1) . The steps in the pathway provide cells with the capacity to selectively eliminate specific proteins under different physiologic conditions. Molecular chaperones, essential for the correct folding function of a number of cellular proteins, also appear to play a key role in the degradation of proteins, as well as in translational modifications such as glycosylation and oligosaccharide trimming. Recently, regulation of endogenous chaperones has been shown to facilitate protein folding and trafficking, enabling newly translated proteins to escape degradation. The National Heart, Lung, and Blood Institute convened a workshop to review the protein folding and quality control pathways operative in rare pulmonary disease, and to identify the research directions that will promote better understanding of how these mechanisms contribute to the molecular pathogenesis of disease and development of novel therapeutic interventions. These issues are likely to be important in understanding the pathogenesis of more common lung diseases as well.



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  		Figure 1. Alternate fates of proteins during biogenesis in the endoplasmic reticulum. Secretory and membrane-associated proteins are initially assembled in the endoplasmic reticulum (ER). Misfolded proteins or mutants that affect interactions of the nascent polypeptide with chaperones may be extracted from the ER and either undergo degradation or accumulate as aggregates. Normal proteins or certain disease intermediates can proceed through the trafficking pathway to the Golgi apparatus. Triangle, squares, and circles represent chaperone proteins.

 
It is becoming increasingly clear that diverse inherited disorders stem from mutations in the genetic material that lead to an abnormal protein structure. These aberrant proteins can be silent until a secondary insult or trigger sets off polymerization, resulting in different fates ranging from the protein being fully formed and nonfunctional, being functional but undergoing premature degradation, or accumulating as aggregates, either inside or outside the cell. Research is starting to link the common threads in the pathogenesis of these diverse diseases related to protein processing and degradation, and to identify early steps in which the processes can be aborted before triggering of a pathogenic cellular response. The pulmonary system is uniquely vulnerable to changes in the environment such as altitude and oxygen tension, inspired toxins, and microorganisms. The interplay between inheritance and environment on protein processing and degradation in the lung is likely to be complex and important.

The cellular response to an abnormal protein in the ER is regulated, complex, and energy-dependent (1), with abnormal proteins being created in a number of ways. Unstable versions appear when nonsense mutations create truncated versions of wild-type proteins. Missense mutations may cause misfolded proteins that are structurally unstable, and proteins can also become denatured as a result of an imposed environmental stress.


   Protein Degradation and Lung Disease
Top
 Introduction
 Protein Degradation and Lung...
Future Directions
 References
 
Many damaged intracellular proteins in mammalian cells are degraded by the proteasome complex through an ATP-dependent process called the ubiquitin-proteasome pathway. This pathway plays a strategic role both in protection against the accumulation of abnormal proteins and in the production of antigenic peptides for presentation to the immune system on MHC class 1 molcules. The ubiquitin carrier enzymes are diverse and tissue- and substrate-specific. A protein marked by the addition of chains of ubiquitin molecules becomes a substrate for the proteasome.

A list of diseases that are the results of defects in intracellular transport, or that cause defects in intracellular transport, is available online at www.traffic.dk (2, 3). Several rare diseases of the respiratory system are caused by mutations in proteins that become dysregulated due to protein misfolding, resulting in their premature degradation in the ER. These include CF, {alpha}1-AT deficiency, and some forms of interstitial lung disease. Model systems that have been explored to delineate the mechanisms regulating these processes include yeast, mammalian cell culture, mice, and humans.

CF transmembrane conductance regulator (CFTR) undergoes rapid ubiquitination and degradation by a proteasome-dependent mechanism (4). Interestingly, the fate of the unstable CFTR appears to vary with the environment. The degradation rate of mutant CFTR is accelerated compared with wild-type CFTR (5). Signaling motifs have been identified in mutant CFTR that influence ER retention and degradation (6). Removal of these trafficking signals overcomes misprocessing of mutant CFTR to escape ER quality control, and promotes mutant CFTR maturation and function at the cell surface. Additionally, some molecular chaperones (Hsp70) associated with the ER have been shown to promote trafficking of the mutant protein, whereas others (Hsc70) promote degradation (7). Recently, CFTR has been shown to traffic through a nonconventional pathway from the ER to the terminal Golgi compartment, bypassing the Golgi apparatus (8). Therapeutic approaches directed toward augmenting this novel pathway offer promise in promoting selective stimulation of mutant CFTR transport to the plasma membrane.

The importance of the selection of the model, and the caution that folding in vitro may not predict folding within intact lungs, cannot be overemphasized. For example, the extent of misprocessing of wild-type CFTR has recently been shown to be dependent on cell type, differentiation state, ambient oxygen, and levels of nitric oxide and reactive species. These studies suggest that common inflammatory diseases of the lung such as asthma, bronchitis, or pulmonary infections may lead to acquired defects in CFTR and in this way contribute to lung pathophysiology (9).

Another disease that results from aberrant protein folding is {alpha}1-AT deficiency. In this deficiency state, emphysema is caused by a loss-of-function mechanism in the form of a deficiency of protease inhibitor in the lung. The liver disease seen in this disorder is caused by a gain-of-function mechanism in the form of cytotoxic effects on the aggregated mutant {alpha}1-AT Z molecule on liver cells. The {alpha}1-AT deficiency strikes 1/1,700 individuals of Northern European ancestry. Homozygotes for the z mutation have 10–15% of normal circulating levels of the protease. The remaining 85% is retained in the liver. Retention of the misfolded aggregate leads to development of micronodular cirrhosis early, and reduction in circulating {alpha}1-AT leads to panlobular basilar emphysema later in life (10, 11).

There is a variable penetrance of liver disease, in that only 10% of subjects homozygous for {alpha}1-AT Z develop clinically significant liver disease. Lomas and colleagues have proposed the hypothesis that the variable penetrance is due to increased childhood infections and fevers that exacerbate the polymerization of {alpha}1-AT Z (10). An alternative explanation for the variable penetrance of liver disease in {alpha}1-AT deficiency comes from studies demonstrating a reduced capacity to dispose of the aggregate {alpha}1-AT Z molecule in cells from the subgroup of {alpha}1-AT–deficient individuals with the severe hepatic phenotype. Recently studies have emphasized the importance of the autophagic response in {alpha}1-AT deficiency, involving the formation of double membrane vesicles to engulf ctyoplasmic and organellar debris. A marked increase in autophagy has been witnessed in liver from {alpha}1-AT–deficient patients in the PiZ mouse model (12).

An important emerging area has been the recent recognition that some interstitial lung disease in adults, as well as chronic interstitial pneumonias of infancy, may involve mutant protein folding (13, 14). The surfactant protein C is vulnerable to mutations that disrupt folding and secretion. Although the SP-C knockout mouse is viable, associations between mutations in the SP-C gene with interstitial lung disease in both infants and adults have been reported (13, 14). In a survey of 45 subjects with interstitial lung disease consisting of desquamative interstitial pneumonitis (DIP), pulmonary alveolar proteinosis (PAP), idiopathic pulmonary fibrosis (IPF), or bronchopulmonary dysplasia (BPD), one third had SP-C mutations, most of which were in the COOH flanking propeptide, suggesting that alteration of misfolding of this region could induce lung disease. A heterozygous exon 5 + 128T->A transversion of SP-C gene has been reported to be associated with familial desquamative and nonspecific interstitial pneumonitis (14).

Regulation of mutant protein folding and fate may be possible pharmacologically. Several groups of proteasome inhibitors have been developed and are now widely used as research tools to study the role of the ubiquitin-proteasome pathway in various cellular processes, and two inhibitors are now in clinical trials for treatment of multiple cancers and stroke (15). Early findings with oral 4-phenylbutyrate (Buphenyl, 4-PBA) in CF have been promising. Short chain fatty acids, 4-PBA and butyrate among them, modulate gene expression by acting as histone deacetylase inhibitors. 4-PBA modulates expression of the heat shock 70-kD protein family, which in turn participates in wild-type and mutant CFTR folding and degradation (7, 16). Early phase I and II clinical trials of 4-BPA in teens and adults with CF show restoration of a modest degree of nasal epithelial chloride transport (17). Continuing safety studies are in progress to detect possible toxicities related to accumulation of aberrant proteins.


   Future Directions
Top
 Introduction
 Protein Degradation and Lung...
 Future Directions
References
 
As a result of the workshop, the following recommendations were formulated for future research directions. Although this workshop focused on protein processing and degradation in rare lung diseases, the issues discussed are likely to be important in understanding pathogenesis of more common lung disorders, including asthma. Specific areas identified as important priorities for future research include studies to identify critical steps, components, and signaling mechanisms operative in lung protein trafficking pathways; the processes by which aberrant proteins are selectively recognized for disposal and in the lung cellular response to accumulation of unfolded proteins; elucidation of the regulation of protein quality control; and exploration of the role of protein misfolding in pathogenesis of lung diseases. Novel therapeutic strategies that can influence these processes in a specific fashion to mitigate or prevent lung disease need to be identified.

  1. More information is needed on the role of chaperones or other agents that promote folding in modulating trafficking of normal and abnormal proteins in heart, lung, and blood disorders. The critical steps in the folding and assembly pathways, the effect of inherited mutations on these processes, and the cellular proteins that mediate and monitor this process need to be identified. How the protein machinery can be redirected to affect and respond to normal processes involving secretory pathways needs to be elucidated.
  2. More information is needed regarding how specific chaperones coordinate the folding and degradation of mutant proteins; the recognition signals of misfolded proteins in the ER; and the molecular machinery that facilitates the folding of the wild-type and mutant forms of the target protein. The components that facilitate intracellular degradation of the mutant target protein need to be identified. How cells respond to the accumulation of misfolded proteins is not understood. Signal transduction mechanisms operative in the cellular response to accumulation of unfolded proteins need to be elucidated. What are the components of ER stress response? How does the cell decide between adaptation and apoptosis?
  3. Events linking intracellular degradation to the unfolded protein response should be defined. Changes or alterations in these components mediate monogenic heart, lung, and blood disorders by mechanisms that need further study.
  4. Chaperone proteins decide the fates of nascent, folding polypeptides, and these interactions are not understood. More needs to be known about how conformational properties impact on processing and degradation of peptides. The critical elements triggering the disease process need to be defined, including the role of environmental stressors on these processes.
  5. More research is needed to determine the levels (or intracellular sites) of protein quality control and the constituents of the multiple checkpoints for quality control. Recognition and degradative mechanisms of misfolded secretory and integral membrane proteins differ. It is not clear whether the proteasome-ubiquitin pathway is the only mechanism responsible for polytopic membrane protein degradation in the ER. The mechanisms of cotranslational versus post-translational quality control need to be defined.
  6. The role of protein misfolding, aggregate formation, and aggresomes in heart, lung, and blood diseases is unknown. How important to disease is protein breakdown into aggresomes? What is the fate of cells expressing aggresomes? Are they protective or toxic for cells? What are the physical, chemical, and cell biologic mechanisms of the intracellular protein aggregation? Do they occur naturally in disease pathogenesis, or are they strictly an artifact of experimental manipulation? Population-based assessments of the prevalence of mutations, the functional analysis of these mutations in vitro to exclude simple polymorphisms, and the generation of relevant in vivo animal models (transgenics) to enhance our understanding of the long-term consequences of expression of mutant proteins is required.
  7. Novel therapeutic strategies that can influence the folding pathways of misfolded polypeptides in vivo need to be identified. It is not known whether pharmacologic modulators can be developed that affect these processes in a specific fashion. Whether the biosynthetic arrest of these different proteins can be overcome is also unknown. Does restoration of the mutant protein to the plasma membrane correct the disease?
  8. Better animal modeling systems expressing mutant protein are needed to explore protein folding and assembly pathways and their alteration in disease, as well to test compounds that correct the folding defect.
  9. High throughput, cell-based assays need to be developed to identify compounds that correct folding defects in lung disease–associated proteins.
  10. Research concerning details of normal protein folding and biogenesis must be supported alongside a disease-oriented approach. Correlation of in vitro and structural data with in vivo observations is critically important. We require a better understanding of which aberrant protein structures or environmental interactions with proteins can trigger an unfolded protein response.


   Acknowledgments
 
This Workshop was supported by the Division of Lung Diseases, National Heart, Lung, and Blood Institute (NHLBI), and the Office of Rare Diseases (ORD) at the National Institutes of Health, Bethesda, Maryland, September 10–11, 2001.


   Footnotes
 
List of Participants: Susan Banks Schlegel, Ph.D.; Michael Beers, M.D.; Jeffrey Brodsky, Ph.D.; Thomas Croxton, M.D., Ph.D.; Douglas Cyr, Ph.D.; Dorothy Berlin Gail, Ph.D.; Alfred Goldberg, Ph.D.; Sandra Guggino, Ph.D.; Henrietta Hyatt-Knorr, Ph.D.; Jan Johansson, M.D., Ph.D.; Randal Kaufman, Ph.D.; Ronald Kopito, Ph.D.; David Lomas, M.D., Ph.D.; Gergely Lukacs, M.D., Ph.D.; Lawrence Nogee, M.D.; Christopher Penland, Ph.D.; David Perlmutter, M.D.; John Riordan, Ph.D.; Richard Sifers, Ph.D.; William Skach, M.D.; Eric Sorscher, M.D.; Philip Thomas, Ph.D.; Timothy Weaver, Ph.D.; William Welch, Ph.D.; and Pamela Zeitlin, M.D., Ph.D.

Received in original form October 1, 2002

Received in final form March 21, 2003


   References
Top
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 Protein Degradation and Lung...
 Future Directions
 References
 

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  3. Aridor, M., and L. A. Hannah. 2001. Traffic jams II: an update of diseases of intracellular transport. Traffic 3:781–790.
  4. Gelman, M. S., E. S. Kannegaard, and R. R. Kopito. 2002. A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 277:11709–11714.[Abstract/Free Full Text]
  5. Sharma, M., M. Benharouga, W. Hu, and G. L. Lukacs. 2001. Confrormational and temperature-sensitive stability defects of the delta F508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments. J. Biol. Chem. 276:8942–8950.[Abstract/Free Full Text]
  6. Chang, X. B., L. Cui, Y. X. Hou, T. J. Jensen, A. A. Aleksandrov, A. Mengos, and J. R. Riordan. 1999. Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis. Mol. Cell 4:137–142.[CrossRef][Medline]
  7. Choo-Kang, L. R., and P. L. Zeitlin. 2001. Induction of HSP70 promotes Delta508 CFTR trafficking. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L58–L68.[Abstract/Free Full Text]
  8. Yoo, J. S., B. D. Moyer, S. Bannykh, H. M. Yoo, J. R. Riordan, and W. E. Blach. 2002. Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway. J. Biol. Chem. 277:11401–11409.[Abstract/Free Full Text]
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