SRC homology 3 (SH3) domains are fundamental modules that enable the assembly of protein complexes through physical interactions with a pool of proline-rich/noncanonical motifs from partner proteins. They are widely studied modular building blocks across all five kingdoms of life and viruses, mediating various biological processes. The SH3 domains are also implicated in the development of human diseases, such as cancer, leukemia, osteoporosis, Alzheimer’s disease, and various infections. A database search of the human proteome reveals the existence of 298 SH3 domains in 221 SH3 domain-containing proteins (SH3DCPs), ranging from 13 to 720 kilodaltons. A phylogenetic analysis of human SH3DCPs based on their multi-domain architecture seems to be the most practical way to classify them functionally, with regard to various physiological pathways. This review further summarizes the achievements made in the classification of SH3 domain functions, their binding specificity, and their significance for various diseases when exploiting SH3 protein modular interactions as drug targets.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein–protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain–PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain–PRM interactions and be utilized as a general approach for other domain–peptide interactions.

We investigated the genetic basis of a previously unclassified congenital vascular anomaly in a patient with multifocal cutaneous vascular tumors. Through genomic analysis, we identified a novel heterozygous de novo germline variant in GA-binding protein-alpha ( GABPA ), an ETS family transcription factor. Histopathologic evaluation demonstrated capillary-venous lesions lacking glucose transporter 1 expression, distinguishing this entity from common infantile hemangioma. Notably, the tumor vasculature exhibited a prominent alpha-SMA-positive perivascular cell layer, with nuclear localized GABPA detected in endothelial and perivascular cells. The GABPA c.509T>G (p.L170R) variant is absent from large population databases, including gnomAD, DeCAF, and RGC-MCPS databases, indicating it is extremely rare. The affected leucine residue is highly conserved across species and located within the Pointed (PNT) domain, a critical region mediating GABPA protein-protein interactions. AlphaMissense predicts this substitution to be highly damaging. Structure-based analysis using an AlphaFold-predicted approach, combined with in silico mutagenesis and molecular interaction analyses, revealed that the L170R substitution introduces new electrostatic interactions while disrupting native hydrophobic contacts within the PNT domain. Functional assessment in zebrafish demonstrated that mosaic stromal expression of GABPA-L170R caused abnormal vascular architecture compared with GABPA-wild-type controls, establishing a direct link between this variant and disrupted vascular development. Collectively, the genetic rarity, evolutionary conservation, predicted structural perturbation, and disruptive effects on vascular development establish GABPA -L170R as likely disease-causing variant. These findings define a new molecular mechanism underlying congenital vascular tumor formation and expand the role for ETS-family transcriptional regulation in human vascular anomalies.

Cellular responses leading to development, proliferation, and differentiation rely on RAF/MEK/ERK signaling that integrates and amplifies signals from various stimuli to cellular downstream responses. The clinical significance of C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, which requires the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF, among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain (CRD; a.a. 136-187), and on the other side, SIRT4 requires predominantly the C-terminus (a.a. 255-314) for full interaction with C-RAF. Interestingly, SIRT4 interacts specifically with C-RAF in a pre-signaling inactive (serine 259 phosphorylated) state. Consistent with this finding, ectopic expression of SIRT4 in HEK293 cells results in upregulation of pS259-C-RAF levels and concomitant reduction of MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, our findings propose another extra-mitochondrial role of SIRT4 and suggest that SIRT4 functions as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its known metabolic tumor suppressor role towards glutamate dehydrogenase and glutamine levels in mitochondria.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs with binding affinities ranging from 0.2 to 125 micromolar out of 300 tested and potential new interactors of SOS1. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3-PRM interactions and be utilized as a general approach for other domain-peptide interactions.

SIRT4 comprises together with SIRT3 and SIRT5 the mitochondrially localized subgroup of sirtuins. SIRT4 regulates via its NAD ⁺ -dependent enzymatic activities mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy). Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl ₂ -induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, within the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl ₂ treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132 prevented CoCl2-induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl ₂ treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms, (i) via mitochondrial autophagy/mitophagy, and (ii) as a separate process via proteasomal degradation within the cytoplasm.