Introduction
In the cellular processes, forkhead-associated (FHA) domains play a crucial role as vital mediators of protein-protein interactions. These versatile domains, composed of 80-100 amino acids and primarily recognized for binding phosphorylated threonine motifs, are ubiquitous across a myriad of organisms, from prokaryotes and eubacteria to complex eukaryotes. Despite their relatively conserved structures, FHA domains exhibit a unique specificity that belies the commonality of phosphorylation events, acting as gatekeepers and signal transducers in essential cellular pathways. This article delves into the enigmatic world of FHA domains, focusing on the roles of two critical FHA-containing proteins, TIFA and TIFAB, in immune signaling and disease.
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Fig. 1 Comparison of structural features of TIFA and TIFAB (Niederkorn M., et al. 2020).
FHA Domains: The Cellular Conductor
FHA domains have the remarkable ability to recognize specific motifs, particularly those involving phosphorylated threonine (Thr) residues on various target proteins. This recognition is akin to a lock-and-key mechanism, where the precise structure of the FHA domain ensures accurate interactions. Found across a multitude of organisms-ranging from prokaryotes and eubacteria to the more complex eukaryotes-these domains often team up with other functional modules, such as kinase domains or RING-fingers, enhancing their ability to recognize and bind substrates. The significant role played by FHA domains in cellular processes like DNA damage response, signal transduction, cell cycle progression, and growth marks them as key players in the regulatory symphony of life.
Interestingly, while the overall folding pattern of FHA domain proteins is well conserved, their interactions exhibit a refined specificity. This specificity arises from distinct residues surrounding the phosphorylated threonine, especially the one located at the +3 position, acting as an additional determinant for precise binding. This fine-tuning ability renders FHA domain proteins highly selective, despite the pervasive occurrence of phosphorylation events in cells. FHA domains, by binding to phosphorylated motifs and shielding them from phosphatases, act as gatekeepers, determining whether and how a phosphorylation signal is processed and recognized. This makes them integral to the regulation of signal transduction pathways in response to cellular stimuli.
TRAF-Interacting Proteins with FHA Domains
In the realm of innate immunity, where rapid and robust responses are crucial to ward off microbial and viral invaders, the discovery of FHA domain-containing proteins has been groundbreaking. Among these, two TRAF-interacting proteins, TIFA and TIFAB, have emerged as pivotal players. These proteins, lacking any intrinsic catalytic activity, leverage their FHA domains to recognize and bind phosphorylated threonine residues on interacting proteins, facilitating essential protein-protein interactions.
TIFA and TIFAB primarily engage with the tumor necrosis factor receptor-associated factor (TRAF) family of proteins. TRAF proteins, essentially ubiquitin ligases, play a central role in transducing extracellular inflammatory signals by synthesizing ubiquitin chains. Among them, TRAF2 and TRAF6 have been pinpointed as key interactors with TIFA, mediating critical pathways like NF-κB signaling. This pathway, crucial for cellular responses to stress, inflammation, and infection, underscores the importance of TIFA and TIFAB in maintaining immune system equilibrium.
The Discovery of TIFA and TIFAB
The journey to discovering TIFA and TIFAB is as fascinating as their roles. TIFA was identified by Kanamori et al. through a mammalian two-hybrid screening approach aimed at finding TRAF2 binding proteins. Located on chromosome 3 in mice and 4q25 in humans, TIFA exhibits high sequence conservation across vertebrates. Not only does TIFA bind to both TRAF2 and TRAF6, but it also potently activates innate immune responses through NF-κB and AP-1 transcription factors.
Shortly thereafter, a homologous search led to the discovery of TIFAB on human chromosome 5q31. While sharing homology with TIFA, TIFAB was shown to exert opposing cellular functions. Unlike many other FHA domain-containing proteins, TIFA and TIFAB do not parade a plethora of supplementary functional domains; their simplicity belies their significant impact.
From Structure to Function: TIFA and TIFAB
Human TIFA, an 184-amino acid protein, exemplifies functional elegance. It includes an FHA domain flanked by short sequences on both ends. TIFA binds directly to TRAF2 and TRAF6, which are integral to inflammatory signaling. Genetic studies revealed that while the FHA domain of TIFA is pivotal for NF-κB activation, the actual binding of TRAF6 is facilitated by residues in the C-terminal region. This intriguing observation led to the discovery that TIFA assembles into higher-order structures, called "TIFAsomes," upon phosphorylation of threonine-9 (Thr-9) in its N-terminus.
These TIFAsomes, enormous signal scaffolds composed of oligomerized TIFA molecules and bound TRAF6 trimers, create an amplifying platform for robust NF-κB signaling. This meticulous assembly allows TIFA to fine-tune immune responses efficiently, ensuring effective signal transmission under sustained pathway stimulation.
Conversely, TIFAB, 161 amino acids long, has piqued interest due to its location within a commonly deleted region of chromosome 5q in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Unlike TIFA, TIFAB suppresses TRAF6-induced NF-κB signaling, revealing divergent roles despite their structural similarities. By interacting with TRAF6, TIFAB inhibits TRAF6 protein levels and downstream inflammatory signaling, acting as a negative regulatory mechanism-a yin to TIFA's yang.
TIFA and TIFAB in Health and Disease
TIFA and TIFAB's dichotomy extends into their involvement in health and disease. As ubiquitously expressed cytoplasmic proteins within hematopoietic cells, TIFA's expression pattern shows a bias towards lymphoid cell fates, hinting at its specialized functions. Emerging research reveals TIFA's significant role in epithelial cells, particularly within the gastrointestinal tract, where it mediates innate immune responses against bacterial infections. The bacteria Helicobacter pylori, known to cause gastric inflammation, triggers TIFA-dependent NF-κB activation, underscoring TIFA's critical role in immune surveillance.
The involvement of TIFA in various diseases highlights its multifaceted nature. From mediating inflammatory responses in endothelial cells under oxidative stress to significant upregulation during hemorrhagic shock, TIFA emerges as a responder to cellular stress. Curiously, in liver cells, TIFA activation during hypoxia-reoxygenation events links it to survival mechanisms, while its role in viral infections, such as enhancing paramyxovirus replication by modulating immune responses, further cements TIFA's importance in pathogen defense.
On the other hand, TIFAB's critical functions in myeloid malignancies, particularly within the context of del(5q) MDS and AML, showcase its role as a stringent regulator of immune signaling. The deletion of TIFAB results in hyperactivation of NF-κB signaling pathways, contributing to hematopoietic progenitor defects and dysplasia. TIFAB also emerges as a critical player in cellular stress responses, interacting with USP15, a deubiquitinating enzyme, to regulate p53 and oxidative stress pathways. This regulation highlights TIFAB's ability to maintain cellular homeostasis, particularly under stress conditions, a function indispensable for preventing aberrant cell proliferation and survival typical in malignancies.
Pioneering the Future of Immune and Cancer Therapies
The intricate roles of TIFA and TIFAB open new avenues in understanding and manipulating immune responses and cancer therapies. Given their ability to modulate essential signaling pathways, these proteins present themselves as promising therapeutic targets, particularly in conditions where immune dysregulation or chronic inflammation plays a pivotal role.
Understanding the structural nuances of TIFA and TIFAB will facilitate the development of novel therapeutics aimed at modulating these proteins' functions. For instance, targeting TIFA's oligomerization or its interaction with TRAF proteins could offer new strategies to enhance immune responses against infections or malignancies. Conversely, strategies focusing on TIFAB's inhibitory role could help mitigate undesired inflammatory responses, particularly in autoimmune diseases or chronic inflammatory conditions.
Conclusion
FHA domain proteins like TIFA and TIFAB stand out as critical regulators of cellular communication, bridging the gap between extracellular signals and cellular responses. Their roles in immune signaling, disease progression, and cellular stress response underscore their importance in maintaining cellular homeostasis. As research continues to unravel their mysteries, these small but mighty proteins promise to be key players in the development of new therapeutic approaches for a range of diseases. The ongoing exploration of their functions and mechanisms will undoubtedly continue to illuminate the complex pathways that govern cellular life, offering hope for advancements in medicine and human health.
References
- Niederkorn M., et al. TIFA and TIFAB: FHA-domain proteins involved in inflammation, hematopoiesis, and disease. Experimental Hematology. 2020, 90: 18-29.
- Durocher D., et al. The molecular basis of FHA domain: phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. Molecular Cell. 2000, 6 (5): 1169-82.
- Hofman K. The FHA domain: a putative nuclear signaling domain found in protein kinases and transcription factors. Trends. Biochem. Sci. 1995, 20: 347-9.
- Matsumura T., et al. TIFAB inhibits TIFA, TRAF-interacting protein with a forkhead-associated domain. Biochemical and Biophysical Research Communications. 2004, 317 (1): 230-4.
- Kanamori M., et al. T2BP, a novel TRAF2 binding protein, can activate NF-κB and AP-1 without TNF stimulation. Biochemical and Biophysical Research Communications. 2002, 290 (3): 1108-13.
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