Comparative functional analysis of proteins containing low-complexity predicted amyloid regions

Dataset compilation

For this study, we downloaded 553,231 protein sequences from SwissProt. To remove redundancy among proteins in the dataset we used CD-HIT (Li & Godzik, 2006) with the aim that no two proteins have ≥40% pairwise sequence identity between them. Any proteins containing non-standard amino acid residues were also removed. Finally we obtained 85,381 protein sequences in total.

Extraction and classification of LCRs

We next identified LCRs in the protein sequences using SEG which uses Shannon’s entropy to search region of low complexity in a protein sequence. During the LCR identification process, SEG collects all possible subsequences of length L having the local sequence complexity ≤K1. All overlapping subsequences having sequence complexity ≤K1 are merged in both directions till the complexity of contig created by overlapping subsequences lie below ≤K2 (Kumari, Kumar & Kumar, 2015). In this work we used default values of SEG (L = 12, K1 = 2.2, K2 = 2.5). Among all LCRs we kept only those which has at least three amino acids. The final dataset had 186,637 LCRs obtained from 59,821 non-redundant proteins. The complete LCR dataset was divided into two sets: Set-I which contained LCRs composed of runs of single amino acid (Harbi, Kumar & Harrison, 2011) and Set-II contained LCRs which were composed of more than one type of amino acids but of similar physico-chemical property. Thus, Set-I has 20 different subsets of LCR, each corresponds to a distinct amino acid. Depending on the functional groups of amino acids, Set-II was also further divided into positive charged (Arg, Lys), negative charged (Glu, Asp), polar (Arg, Lys, Asn, Gln, Asp, Glu) and hydrophobic (Cys, Ile, Leu, Met, Phe, Trp, Val) LCRs.

Functional enrichment

In order to gain insights into the functions, we performed GO-term enrichment analyses on all proteins containing LCRs. This analysis was done using DAVID (Huang Da, Sherman & Lempicki, 2009), which can handle a number of heterogeneous annotation terms (e.g., GO terms, protein domains, pathways and so on) or gene classes and thus helps in visualization of the larger biological picture. For functional analysis we used complete set of SwissProt proteins as background.

Extraction of amyloids in LCRs

Prediction of amyloids in LCRs of human proteome

In order to study the aggregation tendency of LCRs in human proteome, we used human proteome compilation of HPRD (Keshava Prasad et al., 2009). Using default parameters of SEG, we found LCRs in 23,727 proteins out of total 30,046 proteins. Subsequently amyloid regions were predicted in LCR-containing proteins with Waltz.

Functional annotation of human proteins with amyloids in LCRs

Using Protein Analysis THrough Evolutionary Relationships (PANTHER) (Mi, Muruganujan & Thomas, 2013), we analyzed the classes, pathways and functions of human protein which were predicted to have amyloidogenic LCRs. PANTHER does gene annotations on the basis of evolutionary relationships, which were taken from Gene Ontology Reference Genome project.

Compositional trends of LCRs: in general and in amyloids

We first checked prevalent amino acids in LCRs and analyzed whether amino acid composition of LCR affects their aggregation tendency. We did compositional analysis for each LCR sets categorized on the basis of homopolymeric runs, charge and hydrophobicity. The most common homopolymeric runs were polyGln, polyAsn, polySer, polyAla, polyGlu and polyPro (in decreasing order) (Table 1). polyLeu, polyLys, polyAsp, polyGly and polyThr were also found in moderate number. The least preferable homo-repeats were polyVal (13 in number), polyPhe and polyIle (11 in number), polyTyr (seven in number), polyCys (two in number) and polyMet (one in number). We observed total absence of polyTrp LCRs in our dataset (Table 1). The compositional trend analysis on Set-II LCRs revealed that the number of positively charged LCRs was ca. 1/3rd of the negative charged LCRs. The results also showed the number of LCRs composed of polar amino acids was more than hydrophobic amino acids (Table 1).

Prediction of amyloids in LCRs suggests that polyAla, polyPhe, polyLeu, polyAsn, polyGln, polar and hydrophobic amino acids have amyloidogenic capability. Majority of the amyloidogenic LCRs were composed of polyLeu, polyAsn and hydrophobic residues. In contrast, polyCys, polyAsp, polyGlu, polyLys, polyGly, polyHis, polyThr and charged LCRs accounted for a very small fraction of amyloidogenic LCRs with less than 10 residues. The runs of polyMet, polyTrp, polyPro and polyArg were predicted to be completely lacked of amyloidogenic capability (Table 1).

In order to verify our results obtained by prediction of amyloidogenic LCRs, we repeated the analysis on effect of amino acid composition on amyloidogenesis in Data II, which had only experimentally verified amyloidogenic LCRs. The results revealed Gln and Asn as the most abundant; Ala, Gly and Ser as moderate; and Cys and Trp as the least preferred (Fig. 1). This observation was inline with our earlier observation on predicted amyloids in LCRs. We also identified two polar LCRs and one hydrophobic LCR in Data II.

Length analysis of amyloids

In an attempt to investigate whether the amyloids have size variation, we also analyzed the length of amyloidogenic LCRs. In Data I, the homopolymeric runs, predicted to form amyloids, ranged between 3 and 6 AA (Fig. 2). The maximum length of amyloid forming polyLeu runs was 7 AA and polyAsn was 4 AA. For hydrophobic LCRs, shorter amyloids of length 3–11 predominated whereas the length of amyloidogenic polar LCRs were 3–4. We found that hydrophobic LCRs had the longest stretch (18 AA). As the longer hydrophobic stretches are known to be toxic (Dorsman et al., 2002; Oma et al., 2004), this indicates the toxic nature of the amyloidogenic LCRs.

In Data II, which contained only experimentally proven amyloidogenic LCRs, the length of LCRs composed of polar amino acid ranged between 8 and 9 and for hydrophobic LCR, it was 16. This was inline with our observation with Data I.

Functional enrichment of LCRs

To study the functions in which proteins having amyloidogenic LCRs are involved, we analyzed their BP, molecular functions (MF) and cellular components (CC) abundance in each category of LCR using DAVID (https://david.ncifcrf.gov/). For our analysis we considered only the top five enriched GO-terms.

The result showed MF enrichment only in proteins having runs of His, Arg, Asp, Glu, Asn, Gln, Ser, Thr, Ala, Gly and Pro. We found that these protein subsets are strongly associated with metal ion binding, transition metal ion binding and nucleotide binding (Fig. 3A and Table S1). Both polyAsn and polyGln were involved in similar functions, that is, DNA binding and transcription regulator activity. We observed that runs belonging to charged amino acids were involved in ion and DNA binding but LCRs composed of combination of positive and negative charged amino acids were showing additional functions, that is, protein binding (Fig. 4A and Table S1). Polar LCRs were involved in “DNA binding,” “nucleotide binding,” “ATP binding,” “protein binding” and “metal ion binding.” Perfect repeats of hydrophobic amino acids were not enriched in MF whereas LCRs composed of combination of hydrophobic amino acids were involved in “calcium ion binding,” “hydrolase activity,” “receptor activity,” “serine-type endopeptidase activity” and “G-protein coupled receptor activity” (Table S1).

Under BP category, “regulation of transcription” was the most common GO-term. Interestingly, whereas runs of all amino acids were involved in transcription, the topmost enriched function of proteins with polyGly was translational activity (Fig. 3B and Table S2). We also observed that runs of different amino acids were enriched in unique BP. For example, polyArg were involved in “cell surface receptor linked signal transduction,” polyLys in “RNA biosynthetic process,” polyAsp in “protein localization,” polyGlu in “cellular response to stress,” polyGln in “chromosome organization,” polySer in “phosphate metabolic process,” polyThr in “cell division,” “peptidoglycan metabolic process,” “glycosaminoglycan metabolic process” and “regulation of cell morphogenesis,” polyPro in “cytoskeleton organization” and polyVal in “lipid biosynthetic process” (Fig. 3B and Table S2). “Regulation of transcription, DNA-dependent” was common process in the proteins having runs of positively charged amino acids, polyArg and polyLys. Except GO terms “regulation of transcription, DNA-templated” and “transcription, DNA-templated,” BP of LCRs containing combinations of positively charged and combinations of negatively charged amino acids were completely different, for example, positively charged LCRs were involved in “positive regulation of transcription from RNA polymerase II promoter” whereas negatively charged LCRs were involved in “negative regulation of transcription from RNA polymerase II promoter.” Polar LCRs showed enrichment of “transcription, DNA-templated,” “phosphorylation” and “transport” but hydrophobic LCRs were enriched for “signal transduction,” “proteolysis,” “innate immune response” and “transport” (Fig. 4B and Table S2).

In case of CC, the enriched locations were “nuclear lumen,” “organelle lumen” and “membrane enclosed lumen” (Figs. 3C and 4C; Table S3). However, polyLeu, polyPro and polyVal completely lacked any lumen; their enrichment terms were related to “plasma membrane.”

Functional annotation of amyloidogenic LCRs in human proteins

We also analyzed the broad spectrum of functions for each human protein containing predicted amyloidogenic LCRs in terms of GO slim functional categories viz., BP, MF, CC and functional classes using PANTHER. Under BP category, we noticed the annotation terms “biological regulation,” and “response to stimulus” were common to polyAla, polyPhe, polyLeu and hydrophobic LCR (Figs. S1A–S4A). The GO term “cellular process” was found in proteins with polyAla, polyLeu, polar and hydrophobic LCRs (Figs. S2A–S5A). The processes “localization” and “locomotion” were specific to polyAla, polyLeu and hydrophobic LCR (Figs. S2A–S4A); “developmental process” was specific to polyAla and hydrophobic LCRs (Figs. S2A and S4A) whereas the “immune system process” and “biogenesis” to polyLeu and hydrophobic LCR (Figs. S3A and S4A). The processes “biological adhesion,” “biological regulation” and “reproduction” were exclusive for proteins with hydrophobic LCRs (Fig. S4A).

Molecular functions analysis in each category of LCRs showed involvement in “catalytic activity” of proteins with polyAla, hydrophobic and polar LCRs (Figs. S2B, S4B and S5B). The MF term “receptor activity” was observed in proteins with polyPhe (Fig. S1B), polyLeu (Fig. S3B) and hydrophobic LCR (Fig. S4B) while “binding” was enriched in polyAla, polyLeu and hydrophobic LCR. polyPhe and hydrophobic LCR were also associated with “signal transducer activity.” Two additional MF terms “structural molecule activity” and “transporter activity” were unique to hydrophobic LCR.

Furthermore, the CC analysis of amyloidogenic LCRs containing human protein suggested that polyAla and hydrophobic LCR were common in “cell part,” “macromolecular complex” and “membrane” (Figs. S2C and S4C). The CC GO term “extracellular region” was found in proteins with polyLeu and hydrophobic LCR (Fig. S3C). In hydrophobic LCRs, we found additional components such as “extracellular matrix” and “organelle” (Fig. S4C).

We could not found GO slim BP for proteins containing polyAsn, polyGln and polyVal which most likely occurred because GO slim provides a broad outline of GO contents. We then searched specific terms for the proteins in these categories from PANTHER. We found that proteins with amyloids in polyGln had GO BP terms “ion transport” and “neutrophil degranulation” and for proteins with polyVal the terms were “G-protein coupled receptor signaling pathway,” “neuropeptide signaling pathway,” “female pregnancy” and “hormone metabolic process.” The MF term for polyGln was “nucleic acid binding” and for polyVal these were “G-protein coupled receptor activity,” “neuropeptide Y receptor activity,” “protein binding” and “neuropeptide receptor activity.” We found that GO CC terms for polyGln was “lysosomal membrane,” “microtubule organizing center,” “plasma membrane,” “integral component of membrane,” “specific granule membrane,” “intracellular membrane-bounded organelle,” “extracellular exosome” and “tertiary granule membrane.” Proteins containing amyloids in polyVal were associated to “plasma membrane” and “integral component of plasma membrane.”

PANTHER showed pathways for only polyAla and hydrophobic LCRs. We found that proteins of both the categories were mainly associated to various signaling pathways (Table 2). The proteins forming amyloids in hydrophobic LCRs were also involved in many other pathways and cascades such as plasminogen activating cascade, cadherin signaling pathway, Wnt signaling pathway and Alzheimer disease-presenilin pathway (Table 2).

We found protein class annotation for only the LCRs composed of polyAla, polyLeu, polar and hydrophobic amino acids in human proteome. Proteins containing amyloids in polyAla were kinase, DNA binding protein, and forkhead and homeodomain transcription factor (Table 2). Some of the proteins containing polyLeu were Type I cytokine receptor and Chemokine. Polar LCRs which formed amyloid in human proteins contained non-receptor serine/threonine kinase. In case of hydrophobic LCRs, proteins showed diversified class such as glycosyltransferase, chemokine, protease, receptor, enzyme modulator, signaling molecule and transporter (Table 2).

In Data IIh for validated amyloids in human proteins, we found only hydrophobic LCRs that were involved in amyloid formation. In PANTHER, MF of protein containing this LCR is “protein binding”; BP were “respiratory gaseous exchange” and “cellular protein metabolic process”; and CC were “extracellular region,” “extracellular space,” “endoplasmic reticulum membrane,” “integral component of membrane,” “lamellar body,” “clathrin-coated endocytic vesicle” and “multivesicular body lumen.”