IFNG 139 a.a. Human

What is the 139 a.a. human interferon gamma and how does it differ from other forms?

Human interferon gamma (IFNG) is a critical cytokine in immune regulation. The 139 amino acid form represents a specific variant of human IFNG that plays important roles in immune signaling. The human IFNG protein contains basic amino acid clusters in its carboxyl-terminal region that are similar to heparin-binding consensus sequences found in other proteins. Close examination of the human IFNG amino acid sequence reveals these basic residues contribute to its binding properties and biological functions .

This form is distinguished by specific structural features, particularly its carboxyl-terminal region containing basic amino acid stretches that facilitate binding to proteoglycans and extracellular matrix components. Specifically, residues 127-135 (AKTGKRKRS) have been identified as a key sequence involved in binding to chondroitin-sulfate glycosaminoglycans .

How does the binding capacity of human IFNG 139 a.a. to extracellular matrix affect its biological activity?

The interaction between human IFNG and extracellular matrix (ECM) significantly impacts its biological function and availability. Research has demonstrated that IFNG binds to ECM components with high affinity, showing an apparent Kd of 2×10^-11 mol/L with a maximum binding capacity of 1.6×10^6 IFNG molecules per square millimeter of ECM .

This binding is primarily mediated through chondroitin-sulfate proteoglycans (PGs), as evidenced by the significant reduction in binding following treatment with chondroitinase ABC, which degrades chondroitin-sulfate glycosaminoglycans. Importantly, ECM-bound IFNG demonstrates enhanced effectiveness in inducing the expression of class II major histocompatibility complex (MHC) molecules compared to free IFNG . This suggests that the ECM binding serves not only as a reservoir for IFNG but also potentially modulates its signaling capacity and duration of action in tissues.

What are the primary signaling pathways activated by human IFNG 139 a.a.?

Human IFNG activates multiple signaling cascades that regulate diverse cellular processes. The primary signaling pathway involves the JAK-STAT pathway, specifically utilizing STAT1. The search results indicate that several components of this pathway (IFNAR2, IFNLR1, IL10RB, IRF9, STAT1, STAT2, JAK1, and TYK2) were identified in a genetic screen as important factors in interferon signaling .

Additionally, IFNG has been shown to regulate the mTORC1 and MNK pathways that converge on mRNA translation. It influences the translational machinery by affecting the phosphorylation status of eIF4E via MNK1 and by regulating the activation and lysosomal localization of mTORC1 . These pathways collectively contribute to IFNG's ability to modulate cellular metabolism, protein synthesis, and inflammatory responses in a coordinated manner.

How does IFNG regulate cellular metabolism and mRNA translation in macrophages?

IFNG extensively reprograms cellular metabolism and mRNA translation in macrophages through multiple mechanisms:

• Inhibition of mTORC1 activation: IFNG suppresses mTORC1 activity partly through inducing indoleamine 2,3-dioxygenase (IDO), which depletes tryptophan, an amino acid required for mTORC1 activation .

• MNK-eIF4E pathway regulation: IFNG inhibits the activating phosphorylation of MNK1 and eIF4E, reducing translation of specific mRNAs .

• Genome-wide translational reprogramming: Ribosome profiling has revealed that IFNG selectively alters the translational efficiency of nearly 1,000 genes in human macrophages, with 396 genes showing greater than 2-fold changes .

Through these mechanisms, IFNG bidirectionally regulates translation, increasing the translation of inflammatory mediators (TNF, IL-6, lymphotoxins) while decreasing translation of anti-inflammatory factors (IL-10, HES1). This translational regulation extends to metabolic pathways, with increased translation of genes involved in oxidative phosphorylation and mitochondrial functions, and decreased translation of stress pathway components and amino acid transporters .

This table illustrates the canonical pathways most affected by IFNG at the translational level, highlighting its profound impact on cellular metabolism and protein synthesis machinery .

What are the optimal methods for studying IFNG binding to extracellular matrix components?

When studying IFNG binding to extracellular matrix (ECM) components, researchers should consider the following methodological approaches:

• ECM model preparation: Using basement membrane from human arterial smooth muscle cells (HASMCs) in culture provides an effective in vitro model of ECM. This allows for the study of physiologically relevant binding interactions .

• Radiolabeling approach: 125I-labeled IFNG can be utilized to quantify binding parameters. This technique has revealed an apparent Kd of 2×10^-11 mol/L and defined binding capacity metrics .

• Enzymatic treatments: Pretreatment of ECM with specific enzymes like chondroitinase ABC helps identify the glycosaminoglycan components involved in binding. This approach demonstrated that chondroitin-sulfate glycosaminoglycans are primary binding partners for IFNG .

• Competition assays: Using increasing concentrations of purified glycosaminoglycans (such as chondroitin-6-sulfate) to compete with IFNG binding to ECM helps determine binding specificity .

• Affinity chromatography: Immobilizing IFNG and analyzing the binding of [35S]-labeled chondroitin-sulfate proteoglycans from ECM provides confirmation of specific interactions. Subsequent enzymatic treatment with chondroitinase ABC can verify the nature of the bound components .

• Synthetic peptide analysis: Using synthetic peptides corresponding to specific regions of IFNG (such as residues 127-135) helps identify the amino acid sequences involved in binding interactions .

These methods collectively provide a comprehensive approach to characterizing IFNG-ECM interactions at both qualitative and quantitative levels.

How can researchers accurately measure IFNG degradation by proteases like ADAM17?

To accurately measure IFNG degradation by proteases such as ADAM17, researchers should employ the following methodological approaches:

• Time-course incubation studies: Incubating recombinant IFNG with purified ADAM17 for various time periods (20 minutes to 2 hours) allows for the assessment of degradation kinetics. This approach revealed species-specific differences, with human IFNG being more susceptible to degradation than mouse IFNG .

• Western blot analysis: Monitoring the decrease in intact IFNG band density via western blotting provides a semi-quantitative measure of degradation. This technique has shown that human IFNG is degraded by recombinant human ADAM17 within 20 minutes, while mouse IFNG shows significant degradation only after 2 hours .

• Cross-species comparisons: Testing both human and mouse IFNG with recombinant ADAM17 from both species helps determine whether degradation susceptibility is intrinsic to the IFNG structure or dependent on protease specificity. Research has demonstrated that human IFNG is more susceptible to ADAM17 degradation regardless of the protease's species origin .

• ELISA-based quantification: Using enzyme-linked immunosorbent assays to quantify intact IFNG following protease exposure provides more precise measurements of degradation rates.

• Mass spectrometry: Analyzing the cleavage products generated by ADAM17 digestion of IFNG helps identify specific cleavage sites and better understand the structural basis for degradation susceptibility.

These methodologies provide complementary approaches to characterizing protease-mediated IFNG degradation, offering insights into both the kinetics and mechanisms of this process.

How does IFNG contribute to protection against Cryptosporidium infection?

Human IFNG plays a crucial role in the immune response against Cryptosporidium infection through multiple mechanisms. While IFNG itself is important, research has revealed that Cryptosporidium infection primarily induces a type III interferon response (IFNλ) rather than type I or type II interferons in epithelial cells .

A genome-wide knockout screen identified several components of the interferon signaling pathway (IFNAR2, IFNLR1, IL10RB, IRF9, STAT1, STAT2, JAK1, and TYK2) as essential for protection against Cryptosporidium infection. This type III interferon response was shown to require STAT1 signaling in enterocytes and was dependent on sustained intracellular parasite growth .

The protective mechanism involves pattern recognition receptor TLR3, which mediates the production of IFNλ in response to Cryptosporidium. The resulting IFNλ response was demonstrated to reduce infection burden and protect immunocompromised mice from severe outcomes, including death. This response becomes particularly important in early protection against infection, especially in immunocompromised individuals and malnourished children who are most susceptible to severe cryptosporidiosis .

What is the relationship between IFNG and type III interferons in epithelial immune responses?

The relationship between IFNG (type II interferon) and type III interferons (IFNλ) in epithelial immune responses represents an important area of immunological research:

This relationship highlights the specialized and coordinated nature of interferon responses in epithelial tissues, with each interferon type contributing distinctly to host defense.

How can the translational regulation mechanisms of IFNG be leveraged for therapeutic development?

The translational regulatory mechanisms of IFNG present several opportunities for therapeutic development:

• Targeted modulation of mTORC1 and MNK pathways: IFNG inhibits mTORC1 activation and suppresses the MNK-eIF4E pathway. Developing compounds that selectively mimic these effects could provide therapeutic benefits in conditions where inflammatory protein production needs to be modulated .

• Selective translational regulation: Genome-wide ribosome profiling has shown that IFNG increases translational efficiency of inflammatory mediators while decreasing translation of anti-inflammatory factors. This selective regulation could be exploited to develop therapies that target specific translational control mechanisms rather than broad transcriptional regulation .

• Metabolic reprogramming: IFNG's ability to regulate translation of genes involved in metabolic pathways, particularly oxidative phosphorylation and mitochondrial functions, suggests potential applications in metabolic disorders and cancer, where cellular metabolism plays a crucial role .

• Protection from enzymatic degradation: Understanding IFNG's susceptibility to degradation by proteases like ADAM17 opens avenues for developing protease-resistant IFNG variants with extended bioavailability. Human IFNG is more susceptible to ADAM17 degradation than mouse IFNG, suggesting that structural modifications based on cross-species comparisons might yield more stable therapeutic proteins .

• Enhanced tissue retention via ECM binding: IFNG's binding to extracellular matrix components through its basic amino acid clusters could be exploited to develop IFNG variants with optimized matrix binding properties. This could improve tissue retention and sustained local activity, particularly valuable for conditions requiring localized immunomodulation .

These approaches represent promising directions for developing next-generation therapeutics that harness IFNG's sophisticated regulatory mechanisms rather than simply mimicking or blocking its activity.

What are the implications of IFNG's extracellular matrix binding for tissue-specific immune responses?

The binding of IFNG to extracellular matrix (ECM) components has significant implications for tissue-specific immune responses:

• Spatial regulation of activity: IFNG binding to chondroitin-sulfate proteoglycans in the ECM creates localized reservoirs of this cytokine. This spatial regulation allows for concentrated IFNG activity in specific tissue microenvironments, potentially creating immunological niches with distinct properties .

• Enhanced signaling efficacy: ECM-bound IFNG has been shown to be more effective in inducing class II MHC expression compared to soluble IFNG. This suggests that matrix binding may enhance IFNG's signaling capacity, possibly by facilitating receptor clustering or prolonging receptor engagement .

• Tissue-specific immune memory: The persistence of IFNG bound to ECM could contribute to a form of tissue-specific immune memory, where previous immune responses leave an IFNG "imprint" in the tissue that influences subsequent immune challenges.

• Differential activity based on ECM composition: Since tissues vary in their ECM composition, the binding and activity of IFNG may differ between tissues based on their specific glycosaminoglycan content. Tissues rich in chondroitin-sulfate proteoglycans would likely retain more IFNG, potentially leading to heightened sensitivity to this cytokine .

• Implications for pathological conditions: In diseases characterized by ECM remodeling (such as fibrosis, arthritis, or tumor development), alterations in proteoglycan composition could affect IFNG binding and activity, potentially contributing to disease pathology or resistance to therapy.

• Therapeutic targeting: Understanding the specific binding interactions between IFNG and ECM components opens possibilities for developing engineered IFNG variants with modified binding properties for tissue-targeted immunotherapy.

These implications highlight the importance of considering the ECM microenvironment when studying IFNG function in different tissues and suggest that the ECM-cytokine interaction represents an additional layer of regulation in tissue-specific immune responses.

How can researchers address discrepancies between mRNA and protein levels when studying IFNG-induced responses?

Researchers frequently encounter discrepancies between mRNA and protein expression when studying IFNG-induced responses. These discrepancies can be addressed through the following methodological approaches:

• Recognition of translational regulation: It's essential to recognize that IFNG extensively regulates translation. For example, IFNG was shown to almost completely abrogate TLR2-induced HES1 protein expression while minimally affecting its mRNA levels. This indicates that translational control, rather than transcriptional regulation, is the dominant mechanism for certain genes .

• Ribosome profiling: Implementing ribosome profiling in parallel with RNA sequencing provides a comprehensive view of both transcriptional and translational changes. This technique quantifies ribosome-protected RNA fragments, reflecting the translation rate of corresponding mRNAs, and allows calculation of translational efficiency by dividing the change in RPF reads by the change in mRNA abundance .

• Analysis of signaling pathways affecting translation: Examining the activation status of MNK-eIF4E and mTORC1 pathways helps explain translational regulation. Monitoring phosphorylation of MNK1, eIF4E, and mTORC1 components through immunoblotting can reveal mechanisms underlying discrepancies between mRNA and protein expression .

• Pharmacological interventions: Using specific inhibitors of translational regulators (such as the MNK inhibitor CGP57380) can help determine whether observed discrepancies are due to translational control. If protein expression is suppressed by such inhibitors despite unchanged mRNA levels, translational regulation is likely responsible .

• Time-course experiments: Conducting detailed time-course analyses of both mRNA and protein expression helps identify temporal disconnects. In some cases, protein expression may lag significantly behind mRNA changes, or may follow completely different kinetics due to translational regulation and protein stability differences.

By implementing these approaches, researchers can better interpret seemingly contradictory data and gain deeper insights into the complex regulatory networks governed by IFNG.

What are the important considerations for comparing human and mouse IFNG in experimental models?

When comparing human and mouse IFNG in experimental models, researchers should carefully consider several important factors:

• Differential susceptibility to proteolytic degradation: Human IFNG is more susceptible to degradation by proteases such as ADAM17 compared to mouse IFNG. Experiments have shown that human IFNG can be degraded by recombinant human ADAM17 within 20 minutes, while mouse IFNG shows significant degradation only after 2 hours. This differential susceptibility persists regardless of whether human or mouse ADAM17 is used, indicating that it is an intrinsic property of the IFNG proteins themselves .

• Sequence and structural differences: While human and mouse IFNG share functional similarities, they have important structural differences that affect their binding properties, receptor interactions, and susceptibility to regulation. These differences must be considered when extrapolating findings between species.

• Species-specific signaling contexts: The cellular components involved in IFNG signaling may differ between human and mouse systems. While core pathway elements are conserved, there may be species-specific adaptor proteins, regulatory mechanisms, or feedback loops that influence IFNG activity differently.

• Experimental design adaptations: When designing cross-species experiments, researchers should include appropriate controls to account for species-specific effects. This might include parallel experiments with both human and mouse systems, or using species-matched components (cytokines, cells, and reagents) whenever possible.

• Dosage considerations: Due to differences in stability and potency, equivalent molar concentrations of human and mouse IFNG may not produce comparable biological effects. Dose-response studies should be conducted for both species to establish appropriate comparative dosing.

• Interpretation of cross-species reactivity: While some antibodies and detection systems may recognize both human and mouse IFNG, their affinity and specificity may differ. Validation with species-specific positive and negative controls is essential for accurate interpretation of results.

By carefully considering these factors, researchers can design more robust cross-species studies and more accurately translate findings between mouse models and human applications.

What are the emerging areas of research regarding IFNG's role in translational regulation of immune responses?

Several emerging areas of research are expanding our understanding of IFNG's role in translational regulation of immune responses:

• Selective translational control mechanisms: There is growing interest in how IFNG selectively regulates translation of specific mRNA subsets. Genome-wide ribosome profiling has revealed that IFNG both increases and decreases translational efficiency of different gene sets, suggesting sophisticated regulatory mechanisms beyond global translational suppression .

• Integration with metabolic reprogramming: Research is increasingly focusing on how IFNG-mediated translational regulation intersects with metabolic reprogramming in immune cells. The translational regulation of metabolic enzymes, transporters, and mitochondrial components by IFNG suggests a coordinated program that aligns protein synthesis with metabolic capacity .

• Role of tRNA modifications: IFNG strongly downregulates multiple tRNA synthases at the translational level, suggesting potential effects on tRNA availability and modification. Given that these proteins have various noncanonical functions beyond their aminoacyl transferase activity, this represents an exciting area for further investigation .

• Cell-type specific translational landscapes: While comprehensive translational profiling has been performed in macrophages, understanding how IFNG shapes the translational landscape in other immune and non-immune cell types would provide important insights into its tissue-specific functions.

• Long-term epigenetic and translational memory: Research into how IFNG-induced changes in translational efficiency may contribute to trained immunity or long-term functional reprogramming of immune cells is an emerging area with significant implications for understanding sustained immune responses.

• Therapeutic targeting of translational regulation: Developing approaches to selectively modulate IFNG's translational effects represents a promising avenue for therapeutic intervention in inflammatory and infectious diseases, potentially offering more precise control than targeting IFNG itself.

These research directions hold significant promise for deepening our understanding of IFNG biology and developing novel therapeutic strategies based on its sophisticated regulatory mechanisms.

How might advanced structural studies of IFNG-ECM interactions lead to novel therapeutic approaches?

Advanced structural studies of IFNG-extracellular matrix (ECM) interactions hold significant potential for developing novel therapeutic approaches:

• Structure-guided engineering of matrix-binding properties: Detailed structural characterization of the interaction between IFNG's basic amino acid clusters (particularly residues 127-135, AKTGKRKRS) and chondroitin-sulfate glycosaminoglycans could enable rational design of IFNG variants with enhanced or reduced ECM binding . Such variants could be engineered for improved tissue retention or, conversely, broader distribution depending on therapeutic needs.

• Development of matrix-mimetic delivery systems: Understanding the structural basis of IFNG-ECM interactions could inspire the design of biomaterial delivery systems that mimic natural matrix binding. These systems could provide controlled release of IFNG or related immunomodulators while maintaining their bioactivity.

• Targeting tissue-specific glycosaminoglycan compositions: Different tissues have distinct glycosaminoglycan profiles. Structural studies that define how IFNG interacts with various glycosaminoglycan subtypes could lead to the development of tissue-targeted IFNG variants that preferentially localize to specific anatomical sites.

• Creation of matrix-binding peptide therapeutics: The identification of specific peptide sequences involved in matrix binding (such as AKTGKRKRS) provides a foundation for developing smaller peptide therapeutics that either mimic IFNG's matrix interactions or competitively inhibit endogenous IFNG binding .

• Understanding cooperative binding mechanisms: Advanced structural studies might reveal how IFNG binding to ECM influences its interaction with cellular receptors. This could lead to therapeutic approaches that modulate receptor signaling by targeting the ECM-cytokine-receptor complex rather than individual components.

• Development of diagnostic tools: Structural insights into IFNG-ECM interactions could enable the creation of imaging probes or biosensors that detect matrix-bound IFNG in tissues, potentially serving as biomarkers for inflammatory activity or response to therapy.

These approaches represent promising directions for translating structural insights into novel therapeutic strategies that harness or modulate IFNG's interaction with the extracellular matrix.