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Phafins are PH (Pleckstrin Homology) and FYVE (Fab1, YOTB, Vac1, and EEA1) domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins.

Phafin2, a member of the Phafin family of proteins, contributes to a plethora of cellular activities including autophagy, endosomal cargo transportation, and macropinocytosis. The PH and FYVE domains of Phafin2 play key roles in membrane binding, whereas the C-terminal poly aspartic acid (polyD) motif specifically autoinhibits the PH domain binding to the membrane phosphatidylinositol 3-phosphate (PtdIns3P). Since the Phafin2 FYVE domain also binds PtdIns3P, the role of the polyD motif remains unclear. In this study, bioinformatics tools and resources were employed to determine the concurrence of the PH-FYVE module with the polyD motif among Phafin2 and PH-, FYVE-, or polyD-containing proteins from bacteria to humans. FYVE was found to be an ancient domain of Phafin2 and is related to proteins that are present in both prokaryotes and eukaryotes. Interestingly, the polyD motif only evolved in Phafin2 and PH- or both PH-FYVE-containing proteins in animals. PolyD motifs are absent in PH domain-free FYVE-containing proteins, which usually display cellular trafficking or autophagic functions. Moreover, the prediction of the Phafin2-interacting network indicates that Phafin2 primarily cross-talks with proteins involved in autophagy, protein trafficking, and neuronal function. Taken together, the concurrence of the polyD motif with the PH domain may be associated with complex cellular functions that evolved specifically in animals.

Lysosomal degradation of ubiquitinated transmembrane protein receptors (cargo) relies on the function of Endosomal Sorting Complex Required for Transport (ESCRT) protein complexes. The ESCRT machinery is comprised of five unique oligomeric complexes with distinct functions. Target of Myb1 (TOM1) is an ESCRT protein involved in the initial steps of endosomal cargo sorting. To exert its function, TOM1 associates with ubiquitin moieties on the cargo via its VHS and GAT domains. Several ESCRT proteins, including TOLLIP, Endofin, and Hrs, have been reported to form a complex with TOM1 at early endosomal membrane surfaces, which may potentiate the role of TOM1 in cargo sorting. More recently, it was found that TOM1 is involved in other physiological processes, including autophagy, immune responses, and neuroinflammation, which crosstalk with its endosomal cargo sorting function. Alteration of TOM1 function has emerged as a phosphoinositide-dependent survival mechanism for bacterial infections and cancer progression. Based on current knowledge of TOM1-dependent cellular processes, this review illustrates how TOM1 functions in coordination with an array of protein partners under physiological and pathological scenarios.

Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin alpha (IIb)beta (3) receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin alpha (IIb)beta (3) receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate (PtdIns5P), which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.

Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ~28 kDa with an intensity-average hydrodynamic diameter of ~7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an a/b structure and predicts ~40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.48C. The folding-unfolding curves, obtained using urea- and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native-to-denatured transition. Analysis of these transitions shows that the free energy change for urea- and guanidine hydrochloride-induced Phafin2 denaturation in water is ~4 kcal mol21. PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P-binding domains of the protein in endomembrane compartments.

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.