An Integrative Multitiered Computational Analysis for Better Understanding the Structure and Function of 85 Miniproteins
Authors: Veluri Reethika , Pollin Gareth , Wagenknecht Jessica B. , Urrutia Raul , Zimmermann Michael T. 
Published: February 2025

Background

Miniproteins, defined as polypeptides containing fewer than 50 amino acids, have recently elicited significant interest due to an emerging understanding of their diverse roles in fundamental biological processes. In addition, miniprotein dysregulation underlies human diseases and is a significant focus for biotechnology and drug development. Notably, the human genome project revealed the existence of many novel miniproteins, most of which remain uncharacterized. This study reports an approach for analyzing and scoring previously uncharacterized miniproteins by integrating knowledge from classic sequence-based bioinformatics, computational biophysics, and system biology annotations. Our results demonstrate that these approaches provide novel information on the structure-function relationship of these molecules with a particular focus on their biomedical relevance.

Methods

We identified 85 human miniproteins using a simple multi-tier approach. First, we performed a sequence-based analysis of these proteins using several algorithms to identify regions of structural and functional importance. Protein-protein interactions and gene ontology annotations were used to analyze miniprotein function. Then, we predicted miniprotein three-dimensional structures using AI-based methods and peptide modeling to determine their relative yields for these understudied polymers. Subsequently, we used several computational biophysics methods and structure-based calculations to annotate and evaluate results from both algorithms.

Results

We find several relations between predicted structure and functional properties to assign these proteins to several groups with similar properties. Sequence-based analysis leads us to identify motifs and residues that link structure-to-function for most of these proteins. We suggest novel miniprotein functions, such as thymosin beta proteins regulating the shelterin complex through TERF1 and POT1 interactions, FAM86JP and FAM66E participating in endocytic processes, and BAGE1 influencing chromatin remodeling through interaction with nuclear proteins. Further, known functions of miniproteins, such as STRIT1, STMP1, and SLN, were supported. Finally, structure-based scoring led us to build 3D models that provided complementary information to ontologies. We identify that structural propensity is not strictly dependent on polymer length. In fact, in this dataset, peptide-based algorithms may have advantages over AI-based algorithms for certain groups of miniproteins.

Conclusion

This analytic approach and resulting identification and annotation of miniproteins adds much to what is currently known about miniproteins. Our determination of novel properties of miniproteins bears significant mechanistic and biomedical relevance. We propose novel functions of miniproteins, which expands our understanding of their potential roles in cellular processes. And, we practically identify which sequence and structure-based tools provide the most information, aiding future studies of miniproteins.