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Interstitial lung disease in both children and adults has been linked to mutations in the lung-specific surfactant protein C (SFTPC) gene. Among these, the missense mutation [isoleucine to threonine at codon 73 = human surfactant protein C (hSP-CI73T)] accounts for ∼30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP-CI73T induces lung remodeling and alveolar lipoproteinosis without a substantial Endoplasmic Reticulum (ER) stress response or ER-mediated intrinsic apoptosis. We show here that, in contrast to its wild-type counterpart that is directly routed to lysosomal-like organelles for processing, SP-CI73T is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP-C isoforms. Functionally, cells expressing hSP-CI73T demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP-CI73T through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein-associated diseases and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery.
The hydrophobic human surfactant protein C (hSP-C) secreted into the pulmonary alveolus is a product of a larger bitopic, type II integral membrane precursor protein (proSP-C) (depicted in Figure 1) that undergoes palmitoylation and a series of proteolytic cleavages to yield the 3.7-kDa mature form (1,2). Produced exclusively by alveolar type II cells, mature hSP-C is packaged into specialized lysosomal-like organelles (lamellar bodies) and released via regulated exocytosis together with other surfactant proteins and phospholipids. In the alveolus, SP-C along with another surfactant protein, SP-B, plays a critical role in the modulation of lung mechanics from its direct effects on alveolar surface tension. Through enhancement of the adsorption and spreading of surfactant phospholipids and by maintenance of interfacial film at the air–liquid interface, hSP-C promotes the efficient generation of a surfactant monolayer shown to be critical in the prevention of alveolar collapse at end expiration [for review see (1,2)].
Figure 1. Schematic representation of the hSP-C expression constructs used in this study is shown. Additional constructs including EGFP-C1 vector and ABCA3WT•DsRed were used for negative and positive controls. Four proteolytic cleavages of SP-C propeptide at sites “a to d” occur in alveolar type II cells. In contrast, A549 cells cleave the propeptide only once at site “a”. The cytosolic (NH2) and luminal (COOH) domains are indicated.
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Interstitial lung disease (ILD) represents a diverse group of disorders of largely unknown etiology characterized by variable types and degree of interstitial and alveolar inflammation, fibrosis, and impaired gas exchange. Mutations in genes critical for surfactant metabolism and function are now recognized as one of the etiological causes of ILD (3). Of these, the gene encoding hSP-C (SFTPC) has been seminal in establishing this linkage with multiple reports of both sporadic cases as well as an autosomal dominant inheritance pattern with variable penetrance. Histological phenotypes of mutation-associated SFTPC ILD range from chronic pneumonitis of infancy and nonspecific interstitial pneumonitis (NSIP) in children to usual interstitial pneumonia (UIP) and idiopathic pulmonary fibrosis (IPF) in adults (4,5). The great majority of SFTPC mutations are located in the distal C-terminus of proSP-C termed the BRICHOS domain (6), a region of ∼100 amino acids with sequence and structure homology of proteins most notably found in association with familial neurodegenerative dementias. Using a variety of in vitro models, expression of the BRICHOS mutant hSP-C cDNAs results in intracellular aggregation of misfolded protein, ER stress and subsequent induction of apoptotic cell death (7–10).
In addition to these aggregation-prone hSP-C mutations, a different phenotype is emerging for a second group of missense mutations situated outside the BRICHOS domain, but within the C-terminal region of the propeptide adjacent to the transmembrane domain (6,11). Among these, a heterozygous substitution mutation of threonine for isoleucine at amino acid 73 (hSP-CI73T) of the proprotein accounts for ∼30% (over 40 cases reported) of all hSP-C mutations associated with pulmonary disease (11–17) in both de novo (sporadic) as well as in inherited (autosomal dominant) cases. While symptomatic patients with an I73T mutation show some variability in clinical phenotype and course most likely due to the effects of additional modifier genes and/or environmental interactions (4,5,11,17,18), the associated histological and biochemical abnormalities most often include a pattern of NSIP with alveolar type II cell hyperplasia, cellular thickening of alveolar septa and preserved alveolar architecture, often accompanied by evidence of alveolar accumulation of phospholipids and protein (lipoproteinosis). Analyses of the broncho-alveolar lavage of patients with hSP-CI73T also show accumulation of abnormal C-terminal proSP-C products (11,19,20) although the consequences of their presence toward surfactant function is unknown. Moreover, in contrast to hSP-C BRICHOS mutations, in vitro expression of hSP-CI73T fails to either induce ER stress responses, or activate caspase 4 mediated apoptosis (9) suggestive of a mechanism for cellular dysfunction distinct from those elicited by grossly misfolded BRICHOS proteins.
Given the central role of the alveolar epithelium in surfactant metabolism and the unique consequences for the lung resulting from expression of the hSP-CI73T mutation including disrupted surfactant homeostasis, we hypothesized that the trafficking and processing as well as the functional consequences of hSP-CI73T expression in vitro would be distinct from SP-C BRICHOS mutations. In this study, we demonstrate that unlike either its wild-type counterpart or SP-C BRICHOS mutants, the hSP-CI73T proprotein is initially mistargeted to the plasma membrane and subsequently internalized to early endosomes accompanied by aberrant post-translational processing. Functional assays using H3-labeled dipalmitoylphosphatidylcholine (DPPC), the major surfactant lipid species, demonstrated significant reduction of both lipid uptake and degradation by cells stably expressing hSP-CI73T. Thus, expression of this non-BRICHOS mutant results in a unique cellular phenotype distinct from previously characterized SFTPC mutations.
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The cellular mechanisms underlying the pathogenesis of ILD associated with SFTPC mutations are partly understood. Most of the published reports have focused on a select group of mutations located in the proprotein domain flanking the SP-C C-terminus known as the BRICHOS domain (34) where the unfolded protein response (UPR), ER stress, mitochondrial dysfunction and apoptosis have been implicated (7,9,10). In contrast to these, expression of two different non-BRICHOS mutations (I73T, E66K) of the SFTPC gene produces a distinct clinical as well as cellular phenotype. In vivo, non-BRICHOS mutants produce both interstitial pneumonia and pulmonary lipoproteinosis accompanied by abnormal localization of the SP-C propeptide in EEA-1 vesicles in alveolar type II cells (11,35). Since these reports, the hSP-CI73T mutation is now recognized as the most common SP-C mutation to date (13,14,16,17,36). The present report extends the prior findings by demonstrating that trafficking of the hSP-CI73T mutant is altered by its misrouting to the plasma membrane, subsequent internalization to early endosomes and delayed appearance within lysosomal-like compartments. This misrouting is accompanied by aberrant post-translational processing of the mutant isoform, and these abnormalities collectively result in functional impairment of lipid uptake and degradation. Thus, the hSP-CI73T non-BRICHOS mutation represents a new class of spatially and functionally distinct mutations of proSP-C that is capable of producing chronic lung disease, but is phenotypically different from those of misfolded BRICHOS mutants.
In general, the diversion of a protein from reaching its targeted destination due to missense, nonsense, deletion or frame shift mutations is a common phenomenon that occurs in diseases associated with secretory or transmembrane protein mutations. In many cases, protein misfolding is believed to contribute to the pathophysiology. Best known among these are neurodegenerative diseases including Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. For each, a gain-of-function results from mutations in Huntingtin, beta amyloid, SOD1 and α-synuclein, respectively, and produces ER retention of the misfolded protein leading to the activation of the UPR, ER stress and protein aggregation (37). These cellular responses in turn lead to the subcellular disruption of essential proteins such as chaperones, interfere with degradation of other proteasome substrates and compromise the folding and stability of other proteins that are conformationally challenged to cause the global disruption of cellular homeostasis leading to cell death. We have shown some of these cellular abnormalities also occur in vitro in ILD-associated BRICHOS domain hSP-C mutants, including deletion of the exon 4 transcript of the hSP-C gene (Δexon 4) and a missense mutation that substitutes leucine for glutamine at residue 188 (L188Q) (6,10,38). The absence of mature hSP-C in the bronchoalveolar lavages of patients with the heterozygous expression of these mutations suggests a dominant negative effect by the mutant isoform.
Utilizing multiple experimental techniques, data in the present study demonstrate that the hSP-CI73T mutant propeptide is misrouted to the plasma membrane. In contrast to the BRICHOS mutants, hSP-CI73T fails to induce either a dominant negative effect as some mature SP-C can be detected in patients heterozygous for hSP-CI73T or a classic toxic gain of function as significant amounts of ER stress are lacking (9,35). In this way, hSP-CI73T-induced ILD may be more similar to cytotoxic degenerative diseases such as primary hyperoxaluria type 1 (PH1) disease (39), malonyl-coA decarboxylase (MLYCD) deficiency (40) and hereditary hypophosphatemic rickets with hypercalciuria (HHRH) (41). These diseases all result from misrouting of missense mutants to different subcellular compartments or to the plasma membrane, and in most cases lead to cellular dysfunction. Complementary to this concept, our results suggest expression of the aberrantly targeted mutant hSP-CI73T produces a secondary loss-of-function via disruption of lysosomal degradation processes. Moreover, the functional impairment need not be limited to lipid uptake and degradation, but may also extend to other proteins that share these routing compartments. Aberrant processing intermediates of SP-B have been detected in intra-alveolar surfactant fractions in patients with both hSP-CI73T and hSP-CE66K mutations (11,19,35).
In the present study, we have also detected aberrant EGFP•hSP-CI73T intermediates which likely result from mistargeting-dependent exposure of the mutant isoform to alternative processing events from different or additional protease(s). As shown in Figures 4 and 6, there are two additional bands that are absent in the expression profile of wild-type EGFP•hSP-C isoform. These have also been recently identified both in the culture media of cells stably expressing hSP-CI73T and in the lungs of patients with this mutation (19,28). The first band, migrating faster than the primary translation product, could result from intracellular enzymatic cleavage caused by the propeptide's improper routing to EEA1 vesicles. Alternatively, since the same product was biotinylated (Figure 4C), it is possible that trimming of the C-terminal propeptide at the cell surface by opportunistic extracellular proteases may occur. As shown in Figure 9, since proSP-C is a type II integral transmembrane protein (32,42), direct delivery to the cell surface would result in a membrane spanning orientation whereby the C-terminal region is exposed to the extracellular milieu making alternative potential protease cleavages a spatial possibility. Biochemical studies of patients with hSP-CI73T as well as the E66K mutation support the findings of abnormal processing. Aberrantly processed 11- and 13-kDa C-terminal segments of hSP-C proprotein were identified by immunoblotting of bronchoalveolar lavage fluid of a patient carrying the SP-CI73T mutation (11,35). Mass spectrometry has also shown intra-alveolar accumulation of aberrant C-terminal processing products (19).
Figure 9. Model for aberrant hSP-CI73T trafficking. Schematic representation depicting normal and abnormal sorting of wild-type and mutant isoforms of SP-C. The hSP-CWT gene/cDNA is transcribed into a 0.9-kb mRNA product which is translated to a 197 amino acid (21 kDa) proprotein. Proteolytic cleavages occur during transit through the secretory pathway that include small vesicles, multivesicular bodies and lysosome-like vesicles (LLV). In contrast, hSP-CI73T is misdirected to the plasma membrane via a nonendosomal and likely through the constitutive pathway where it is subsequently internalized and sorted to early endosomes and LLV. N and C depict the N- and C-termini of the proprotein. Hatched arrows imply pathways that may require multiple steps to reach target organelles. (Inset) Topological representation of the bitopic proSP-C with-type II orientation. Mature SP-C (blue), cleavable segments of proSP-C (green), Juxtamembrane palmitoylation sites (arrow), and a C-terminal disulfide-bonding site (arrowhead) are shown.
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In addition to abnormal protein processing, other consequences of hSP-CI73T mistargeting include an accompanied ultra structural dysfunction. Alveolar type II cells are actively involved in the clearance of pulmonary surfactant from the intra-alveolar space as well as recycling [via the multivesicular bodies (MVBs)] of internalized surfactant lipids and proteins back to the lamellar body (43,44). Interestingly, the lamellar body itself exhibits characteristics of a lysosomal-like secretory organelle (45). Consequently, the misrouting of proSP-CI73T to the cell surface and its subsequent internalization not only affects the lysosomal degradation pathway, but may also disrupt the MVB/lamellar body recycling system including docking and fusion processes of exocytosis. Thus, the observation of abnormal dense core organelles and poorly developed lamellar bodies in alveolar type II cells of patients with hSP-CI73T(11,17) is likely the product of both disrupted lysosomes and a functionally compromised endosome/MVB/lamellar body recycling system. Moreover, since patients with SP-CI73Thave alveolar lipoproteinosis, the increased steady-state level of surfactant reflects impaired endocytosis (supported by the present study) and also implies enhanced secretion.
To address this functionally, we modeled the effect of hSP-CI73T expression on clearance of lipid components using stably transfected cell lines. The reduced phospholipid uptake and degradation demonstrated in Figure 8 conforms to disorders associated with defects in clearance and recycling of surfactants from lung. Under normal conditions, both type II cells and alveolar macrophages contribute to surfactant clearance from the distal airways. Previously, in both humans and transgenic mice, defects in granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling either by ligand- or receptor-inhibition (by neutralizing antibodies or mutations) have been associated with disruption of surfactant homeostasis where the bronchoalveolar lavages of patients lacking GM-CSF activity have revealed pulmonary alveolar lipoproteinosis (46–48). Likewise, targeted deletion in mice of the acid-independent phospholipase A2(aiPLA2), a lysosomal enzyme that degrades endocytosed surfactant DPPC in alveolar type II cells, results in the time-dependent accumulation of both total surfactant phospholipid [mainly disaturated phosphatidylcholine (PC) in bronchoalveolar lavage, lung homogenate and lung lamellar bodies] (49). Therefore, the intra-alveolar accumulation of lipids and protein in patients with hSP-CI73T mutation could directly be attributed to a malfunction in type II cell-dependent surfactant clearance and recycling and suggests both cell types are important for surfactant homeostasis.
In summary, we report phenotypic and biochemical characterization of a non-BRICHOS mutation of the SFTPC gene associated with interstitial pneumonia and alveolar lipoproteinosis. We have determined aberrant protein trafficking and subcellular localization (Figure 9) of this propeptide leading to abnormal protein processing. The nature of the mutation appears to dictate an alternative biosynthetic route taken by the mutant propeptide, which is distinctly different in both spatial and temporal phases from those observed within BRICHOS folding mutants. The acquired impairment of lipid uptake and degradation by cells expressing the mutant propeptide support data from earlier reports of patients with abnormal intra-alveolar contents of lipids and proteins, including increased surfactant protein A content and aberrantly processed surfactant proteins B and C. In addition, this functional impairment points to a novel concept where a protein that is not functionally associated with lysosomes can compromise the lysosomal-dependent degradation process. Understanding molecular mechanisms underlying the etiology and evolution of these abnormalities will provide a pertinent perspective to disease progression, and may offer insights into the development of new therapeutic strategies tailored to the specific subcellular deficits and pathways induced by such mistargeted proteins.