Fungal Immunomodulatory Proteins (FIPs) are small, heat stable macromolecules, usually 110–114 amino acids long with molecular weight ∼ 13 kDa. They have been mostly isolated and identified from medicinal or edible higher fungal organisms sharing functional and structural characteristics. FIPs are rich in valine (Val) and asparagine (Asn) residues but they lack cysteine (Cys), histidine (His) and methionine (Met) (Li et al., 2011). According to their conserved structures, FIPs can be divided into five subgroups: Flamulina velutipes (Fve) −type FIPs, Cerato-type FIPs, Poria cocos protein − PCP-like FIPs, Tremella fuciformis protein TFP-like FIPs, and unclassified FIPs. Structurally, they mostly exist as homodimers, and each subunit contains an N-terminal α-helix dimerization domain and a C-terminal fibronectin III domain (FNIII). The N-terminal domain starts with an N-terminal α-helix, which is followed by a β-sheet or a stretch of random coil, which is essential for the formation and stability of the dimeric structure and connects the N-terminal with the C-terminal portions. Generally, FIPs are stabilized thanks to hydrophobic interactions inside the N-terminal, while the C-terminal portion forms an immunoglobulin-like β-sheet fold (7 β-sheets) connected via loop (Paaventhan et al., 2003). Additionally, it was demonstrated that the FIPs protein family indeed display sequence similarity with the family of immunoglobulins (Paaventhan et al., 2003, Pushparajah et al., 2016, Tanaka et al., 1989).

Research revealed that FIPs pose valuable therapeutic and pharmaceutical biological activities. FIPs were proposed to influence immune responses by modulating cytokine activity, balancing pro-inflammatory and anti-inflammatory pathways (Lin et al., 2023). Recent studies suggest that FIPs can also stimulate mitochondrial activity within immune cells, indirectly impacting OXPHOS as a part of cellular metabolism (Lin et al., 2021, Saccone et al., 2006, Tragni et al., 2022b). Since immune cells, especially activated ones, rely on high mitochondrial activity and ATP production through OXPHOS (Lin et al., 2021, Lin et al., 2023, Todisco et al., 2023, Tragni et al., 2022b), FIPs may play a role in enhancing mitochondrial efficiency in such contexts.

More in general, the ability of FIPs to influence immune cells, make them an attractive target to be investigated for their antitumor and anti-/pro-inflammatory (immunomodulatory) properties. It was observed that immune cells activation can be monitored by observing T-cell activation, macrophage modulation or antigen-presenting cells (APC) activation (Li et al., 2019a). In this regard, Hsu et al. investigated Ganoderma lucidum FIP (LZ-8) regulation on IL-2 transcriptional expression on T-cells, and their results indicated that rLZ-8 could up-regulate this expression by mediating signal-transduction pathways via protein kinase-dependent signaling (Hsu et al., 2008). Moreover, Ganoderma sinensis FIP (FIP-gsi) was able to trigger transcriptional expression of TNF-α and IL-4, IL-3, IL-2, IL-2 receptor and IFN-γ (Li et al., 2010). Conversely, Flammulina velutipes FIP (FIP-fve) exercised the opposite effect by significantly decreasing the secretion of IL-17, IL-13, IL-5 and IL-4 in airway inflammation mice models (Chu et al., 2017, Lee et al., 2013).

Remarkably, it was observed that FIPs share similarities in protein sequences, but FIPs from different species might exhibit diverse biological activities. For example, LZ-8 exhibits potent antitumor effects on HL60 cells, while Fip-fve lacks such activity, despite an 81 % sequence similarity between them (Huang et al., 2009). In vitro and in vivo studies indicate direct and indirect effects on cancer cells as well. The possible antitumor effect is hypothesized to depend on direct cytotoxicity towards cancer cells/anti-proliferative mechanism/induction of apoptosis (Hsin et al., 2020), which are fundamental processes of programmed cell death to regulate the tissue and organ homeostasis. FIPs are of great interest, particularly because they do not cause side effects in allergy treatments or when used as an adjunct to combat drug-resistant tumor cells, as indicated by recent research findings (Lin et al., 2011). Exploring the simultaneous administration of drugs with Fve-type FIPs is also significant, given observations suggesting that certain Fve-type FIPs can maintain their biological activity even when orally administered (Chang et al., 2007, Huang et al., 2014, Tong et al., 2008). This suggests that they may resist digestion or that essential domains responsible for their potency remain intact upon reaching the intestinal tract (Ou et al., 2009). These characteristics define a favorable pharmacokinetic profile for FIPs.

Six different FIP crystallized structures are available in the Protein Data Bank with specific reference to 3f3h.pdb (Huang et al., 2009), 3kcw.pdb (to be published), 7wdm.pdb (to be published), 7wdl.pdb (Liu et al., 2022), 8go7.pdb (Liu et al., 2023), 1osy.pdb (Paaventhan et al., 2003). The structures mentioned were crystallized with the aim of contributing to the identification of crucial motifs responsible for the diverging observed biological activities. Additionally, the structures of FIPs 1osy.pdb (Paaventhan et al., 2003) and 3f3h.pdb (Huang et al., 2009) allowed to investigate their thermal stability and how the glycosylation process can improve either the thermostability or digestion resistance, to support the suitability of FIPs for oral administration (Liu et al., 2023).

While there are numerous reports highlighting the pharmacological potential of FIPs, their precise mechanisms remain unclear (Li et al., 2019b, Liu et al., 2020). To address this gap, we have decided to perform a 3D comparative analysis between FIPs and structurally related proteins, involved in the immune response by using molecular modeling. This will allow us to shed light on possible FIPs sequence/structural/functional relationships and potential interactions with other immunomodulatory actors, paving the way for the design and development of FIP derivatives as therapeutic agents for human immune diseases.