Exploring the context of diacidic motif DE as a signal for unconventional protein secretion in eukaryotic proteins

Abstract

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid (‘X’ in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

Early Bioinformatic Implication of Triacidic Amino Acid Motifs in Autophagy-Dependent Unconventional Secretion of Mammalian Proteins

“…Of these two statistically significant observations, the triacidic motif, by coincidence, happens to be an extension of the observation of the diacidic motif (Cruz-Garcia et al, 2017) and our earlier attempt to find the context in which the diacidic motif appears (Padmanabhan et al, 2018). In fact, a quick reanalysis of the multiple sequence alignments from the homologs of SOD1, Acb1 (Cruz-Garcia et al, 2017) by focusing on the mammalian sequences alone shows that they all have a common triacidic motif (Figure 1).…”

“…Thus, it appears that triacidic or KKX amino acid motifs in the proximity of LIR and/or phosphorylatable amino acids may play a significant role in triggering the unconventional secretion. This result was also validated in the independently curated dataset of unconventionally secreted proteins from other mammalian cells (Padmanabhan et al, 2018), where among the 26 mammalian proteins that are secreted unconventionally, 5 of them had triacidic motifs within a 5 amino acid proximity of LIR. 9 of the remaining proteins where there was no sequence proximity, but the structures were available, had LIR motifs within 10 Å of the triacidic motif, and three other structures had them within 15 Å.…”

“…One of the early indications for what may be a possible signal in this fascinating unconventional secretion process, has only recently been discovered. The discovery of the diacidic motif, DE as the signal for UCPS of SOD1 (Cruz-Garcia et al, 2017) along with the context dependence of the presence of this motif in proximity with the charged, unstructured amino acids (Padmanabhan et al, 2018) might provide some clues. Similarly, motif-1 of the interleukin family is demonstrated to help in driving the unconventional secretion process (Zhang et al, 2020).…”

A systematic review of Sec24 cargo interactome

“…However, presence of existing ER export motifs should not be solely used to predict export of proteins. For instance, a study identified 100 cargoes that contained putative diacidic motifs and proposed that the functional ER export diacidic motifs seem to appear in a region with ordered structure and higher hydrophobicity 95 . The importance of the dihydrophobic nature, rather than the specific residues, of the ER exit signal 'FF' in ERGIC‐53 was found by replacing the di‐phenylalanine 'FF' motif by di‐tyrosine 'YY', di‐valine 'VV', di‐isoleucine 'II' or di‐leucine 'LL' which mediated transport as efficiently if not more than the 'FF' motif 80,96 .…”

Facets of Autophagy Based Unconventional Protein Secretion–The Road Less Traveled

“…Likewise, the specific phosphorylation of the small heat shock protein, α-Crystallin B at the Ser59 residue enhances autophagy mediated secretion ( D’Agostino et al, 2019 ). The discovery of the diacidic motif, DE as the signal for UCPS ( Cruz-Garcia et al, 2017 ) along with the context dependence of the presence of this motif in proximity with the LIR motif ( Padmanabhan et al, 2018 ) might provide some clues. With the DE as the UCPS export signal, the LIR containing proteins possess specific membrane associated receptors and the cells might use this in combination for the type III secretion.…”