Journal of Molecular Biology
Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations
Graphical Abstract
Introduction
Secretory and membrane proteins represent more than a third of all proteins in a cell [1]. During their biogenesis, these proteins are transported to different cellular compartments or secreted outside of the cells. Numerous human diseases are associated with protein transport defects [2], [3], [4]. Many secretory proteins are synthesized as precursors with N-terminal signal sequences. When a signal sequence of a secretory protein emerges from the ribosome exit tunnel, it is co-translationally recognized by the signal recognition particle (SRP) [5], [6], [7], [8], [9]. Formation of a SRP–ribosome–nascent chain complex leads to its targeting to SRP receptor in the endoplasmic reticulum (ER) membrane [10]. Finally, the nascent peptide with its signal sequence is transferred to the Sec61 translocon and a secretory protein is co-translationally translocated into the ER lumen, the signal sequence is cleaved off by the signal peptidase at the luminal side of the ER membrane, and matured proteins are transported further through Golgi outside the cell. These processes are relatively well studied and have been reviewed in detail [11], [12], [13], [14].
Signal sequence recognition by SRP is the first and the most important step in targeting of the SRP–ribosome–nascent chain complex to the ER membrane for translocation. Although signal sequences do not have strong amino acid homology, majority of them have similar features and structural organization: a positively charged N-terminal n-region, a hydrophobic core (h-region), and a C-terminal c-region containing cleavage site for a signal peptidase [15], [16] (Fig. 1a). Signal sequence integrity is important for protein targeting and translocation through a membrane in prokaryotes and eukaryotes [17], [18], [19], [20], [21]. A number of naturally occurring mutations in signal sequences have been found, many of them associated with human disease [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]. Currently accepted mechanisms for these diseases are connected with defects in the transport or processing of the mutated proteins.
Recently, we demonstrated the existence of a novel mechanism for quality control of secretory proteins, named regulation of aberrant protein production (RAPP) [43]. This is a unique quality control pathway among others—it senses interactions of nascent chains during synthesis and degrades mRNAs of proteins that have lost these important interactions [44]. We demonstrated that the pathway degrades mRNAs of model secretory proteins with dramatic deletions in signal sequences when SRP is not able to recognize the altered signal sequences [43]. Although the existence of the pathway is currently established, many details of the mechanism remain unknown. Only one natural substrate of the RAPP pathway is currently identified—granulin with mutations in signal sequence [45], [46]. Here, we reveal that the RAPP pathway is involved in quality control of many secretory proteins with disease-associated mutations. We demonstrate on the example of a number of secretory proteins with disease-associated mutations in their signal sequences that the molecular mechanism is different from the current dogma and connected with stability of the mutated protein mRNAs instead of protein transport defects. We show that many disease-associated mutations lead to degradation of the mutant protein mRNAs, implicating the RAPP pathway as a molecular mechanism of these diseases.
Section snippets
Mutations in the signal sequences of secretory proteins are associated with human diseases
We conducted a literature search for proteins with disease-associated mutations in their signal sequences. Some of these proteins and disease-associated mutations are presented in Table 1. These proteins and the diseases represent a quite diverse group—the proteins have different functions, molecular weight, and signal sequence length. However, many disease-associated mutations have common features: they are located in the hydrophobic core of the signal sequences and led to significant changes
Discussion
SRP-dependent protein targeting initiates the major pathway for transport of secretory and membrane proteins. The process involves co-translational recognition of signal sequences by SRP. A number of secretory proteins with disease-associated mutations in the signal sequences were examined in this study (Table 1). Defects in the transport of mutant proteins through the secretory pathway are often assumed to underlie the diseases. Here we demonstrate that the molecular mechanism behind the
Cloning, mutagenesis, DNA, and RNA techniques
The clones containing ORFs of the genes LIPA (BC012287.1), COL10A1 (NM_000493.3), PRSS1 (NM_002769.4), SERPINE1 (NM_000602.4), LHB (NM_000894.2), TGFB1 (NM_000660.4), PTH (NM_000315.2), NDP (NM_000266.3), SERPINA7 (NM_000354.5), UGT1A1 (NM_000463.2), and CTSK (NM_000396.3) were obtained from Life Technologies, and CTLA4 (NM_005214.4) and AGA (BC012392.1) were obtained from Sino Biologicals. All cDNAs were cloned into pCS2 vector under control of the CMV promoter. Site-directed mutagenesis was
Acknowledgments
Authors thank Dan Webster (Texas Tech University Health Sciences Center) for a gift of anti-tubulin rabbit polyclonal antibody. The research was supported by the Start-up funds from Texas Tech University Health Sciences Center and by a grant from the National Institutes of Health (R03NS102645) to A.L.K. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Author contributions: A.L.K. and Z.N.K.
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