Microprotein

A microprotein (miP) is a small protein encoded from a small open reading frame (sORF),¹ also known as sORF-encoded protein (SEP). They are a class of protein with a single protein domain. They are related to multidomain proteins.² Microproteins regulate larger multidomain proteins at the post-translational level.³ Microproteins are analogous to microRNAs (miRNAs) and heterodimerize with their targets causing dominant and negative effects.⁴ In animals and plants, microproteins influence many biological processes.² Because of their dominant effects on their targets, microproteins are currently under study for use in biotechnology.² In humans, they are associated with genetic diseases and cancers, and are called peptideins.⁵

The first miP was discovered during a research in the early 1990s on genes for basic helix–loop–helix (bHLH) transcription factors from a murine erythroleukaemia cell cDNA library.³ The protein was an inhibitor of DNA binding (ID protein), and negatively regulated the transcription factor complex.³ The protein was 16 kDa and consisted of a helix-loop-helix (HLH) domain.² The microprotein formed bHLH/HLH heterodimers that disrupted the functional basic helix–loop–helix (bHLH) homodimers.²

The first plant microprotein discovered was the LITTLE ZIPPER (ZPR) protein.² The LITTLE ZIPPER protein contains a leucine zipper domain, but lacks the domains required for DNA binding and transcription activation.² Thus, LITTLE ZIPPER protein is analogous to the ID protein.² Although not all proteins are small, in 2011, this class of protein was given the name microproteins because their negative regulatory actions are similar to those of miRNAs.³

The ID protein or proteins similar to ID are found in all animals.³ Plant microproteins are only found in higher orders.³ However, the homeodomain transcription factors that belong to the three-amino-acid loop-extension (TALE) family are targets of microproteins, and these homeodomain proteins are conserved in animals, plants, and fungi.³

Microproteins generally feature a single protein domain.²⁴ The active form is translated from smORF.¹ smORFs can be less than 100 codons.¹ However, not all microproteins are small, and the name was given because of the analogy to miRNAs.³ Despite their short length, microproteins have been shown to a limited but diverse set of structural folds (including predominantly α-helical and transmembrane-helical structures), but many candidates also show substantial intrinsic disorder; experimentally determined microprotein structures have been solved using approaches including X-ray crystallography, cryo-electron microscopy, and NMR.⁶ A computational survey of predicted structures for 44 microproteins reported broadly similar structural characteristics across the set and comparatively few predicted small-molecule ligand-binding sites.⁷ Because many structure/disorder predictors are trained primarily on longer “classical” proteins, dedicated workflows and precautions have been proposed for reliable structure and disorder prediction for microproteins.⁸

Microproteins function as post-translational regulators.³ Microproteins disrupt the formation of heterodimeric, homodimeric, or multimeric complexes.⁴ Furthermore, microproteins can interact with any protein that requires functional dimers to function normally.³ The primary targets are transcription factors that bind to DNA as dimers.⁹³ Microproteins regulate these complexes by creating homotypic dimers with the targets and inhibit protein complex function.³ The two types of miP inhibitions are: homotypic miP inhibition and heterotypic miP inhibition.⁴ In homotypic miP inhibition, microproteins interact with proteins with similar protein-protein interaction (PPI) domain.⁴ In heterotypic miP inhibition, microproteins interact with proteins with different but compatible PPI domain.⁴ In both types of inhibition, microproteins interfere and prevent the PPI domains from interacting with their normal proteins.⁴