Tryptophan Environment Research Articles

Novel unfolding sequence of banana lectin: Folded, unfolded and natively unfolded-like monomeric states in guanidine hydrochloride

The sequence of unfolding events of dimeric banana lectin (Banlec), as induced by guanidine hydrochloride (GdnHCl), has been investigated by size-exclusion HPLC, fluorescence, far-UV CD, low temperature phosphorescence and selective chemical modification. 8-Anilino-1-naphthalenesulfonate (ANS) binding indicates a structured unfolding intermediate which has been characterized as dissociated monomer by size-exclusion chromatography. Interestingly, the unfolding elution pattern reveals two distinct unfolded states. One is a usual random coil. The other represents a novel species having elution behavior and structural compactness (Stokes radius) similar to dissociated monomer but showing no regular secondary structure as determined by far-UV CD, thus resembling a natively unfolded state. N-Bromosuccinimide (NBS) oxidation shows that single tryptophan residue remains unmodified in dissociated monomer intermediate while the same is oxidized in natively unfolded-like species. Such difference in tryptophan environment in these species is supported by acrylamide quenching studies, and phosphorescence results at 77K which show a blue-shift of (0,0) band from 414.8nm to 409.2nm. The present results reveal subtlety of structural characteristics of unfolded states of Banlec in GdnHCl, which provide important insight in protein unfolding reaction.

Thermally stable harpin, HrpZPss is sensitive to chemical denaturants: Probing tryptophan environment, chemical and thermal unfolding by fluorescence spectroscopy

Harpins - a group of proteins that elicit hypersensitive response (HR) in non-host plants - are secreted by certain Gram-negative plant pathogenic bacteria upon interaction with the plant. In the present study, the microenvironment and solvent accessibility of the sole tryptophan residue (Trp-167) in harpin HrpZPss, secreted by Pseudomonas syringae pv. syringae, have been characterized by fluorescence spectroscopic studies. Emission λmax of the native protein at 328 nm indicates that Trp-167 is buried in a hydrophobic region in the interior of the protein matrix. Significant quenching (53%) was seen with the neutral quencher, acrylamide at 0.5 M concentration, whereas quenching by ionic quenchers, I(-) (~10%) and Cs(+) (negligible) was considerably lower. In the presence of 6.0 M guanidine hydrochloride (GdnHCl) the emission λmax shifted to 350.5 nm, and quenching by both neutral and ionic quenchers increased significantly, suggesting complete exposure of the indole side chain to the aqueous medium. Fluorescence studies on the thermal unfolding of HrpZPss are fully consistent with a complex thermal unfolding process and high thermal stability of this protein, inferred from previous differential scanning calorimetric and dynamic light scattering studies. However, the protein exhibits low resistance to chemical denaturants, with 50% unfolding seen in the presence of 1.77 M GdnHCl or 3.59 M urea. The ratio of m value, determined from linear extrapolation model, for GdnHCl and urea-induced unfolding was 1.8 and suggests the presence of hydrophobic interactions, which could possibly involve leucine zipper-like helical regions on the surface of the protein.

Denaturation of bovine spleen galectin-1 in guanidine hydrochloride and fluoroalcohols: structural characterization and implications for protein folding

Guanidine hydrochloride (GdnHCl)-induced unfolding of bovine spleen galectin-1 (Gal-1) exhibits three-state mechanism involving exclusive, structured tertiary monomer in 0.5 M GdnHCl. Gal-1 has one tryptophan residue (Trp 68) per subunit. Phosphorescence spectra of both Gal-1 dimer and intermediate monomer at 77 K show single (0,0) band at 405.6 nm, characteristics of free tryptophan environment as of N-acetyl-l-tryptophanamide. Unfolded Gal-1 in 4 M GdnHCl gives (0,0) band at longer wavelength (408.6 nm) signifying that Trp 68 is less solvent exposed, being localized in an environment of residual structure. Trifluoroethanol (TFE)- and hexafluoroisopropanol (HFIP)-induced structural changes of Gal-1 dimer and monomer have been investigated by far-UV CD and FTIR. CD results show reversible nature of β-sheet to α-helix transition, with 30% helix in 80% TFE or 40% HFIP for Gal-1 dimer. Temperature-dependent studies show that induced helix entails reduced thermal stability. FTIR results reveal partial β-sheet to α-helix conversion but with quantitative yield. At intermediate TFE concentration, both Gal-1 dimer and monomer aggregate as evidenced by FTIR band at ∼1617/cm, however, the onset of aggregation and stability of aggregates for monomer differ from those of dimer. The results may provide important insights into perturbed folding problem of Gal-1.

Molten Globule of Hemoglobin Proceeds into Aggregates and Advanced Glycated End Products

Conformational alterations of bovine hemoglobin (Hb) upon sequential addition of glyoxal over a range of 0–90% v/v were investigated. At 20% v/v glyoxal, molten globule (MG) state of Hb was observed by altered tryptophan fluorescence, high ANS binding, existence of intact heme, native-like secondary structure as depicted by far-UV circular dichroism (CD) and ATR-FTIR spectra as well as loss in tertiary structure as confirmed by near-UV CD spectra. In addition, size exclusion chromatography analysis depicted that MG state at 20% v/v glyoxal corresponded to expanded pre-dissociated dimers. Aggregates of Hb were detected at 70% v/v glyoxal. These aggregates of Hb had altered tryptophan environment, low ANS binding, exposed heme, increased β-sheet secondary structure, loss in tertiary structure, enhanced thioflavin T (ThT) fluorescence and red shifted Congo Red (CR) absorbance. On incubating Hb with 30% v/v glyoxal for 0–20 days, advanced glycation end products (AGEs) were detected on day 20. These AGEs were characterised by enhanced tryptophan fluorescence at 450 nm, exposure of heme, increase in intermolecular β-sheets, enhanced ThT fluorescence and red shift in CR absorbance. Comet assay revealed aggregates and AGEs to be genotoxic in nature. Scanning electron microscopy confirmed the amorphous structure of aggregates and branched fibrils of AGEs. The transformation of α-helix to β-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington's.

Detection and analysis of protofibrils and fibrils of hemoglobin: Implications for the pathogenesis and cure of heme loss related maladies

TFE induces structural alterations of proteins similar to the lipid environment of biological membranes, implicating these studies worthy of analyzing protein conformation in membranes such as red blood cells (RBCs). Heme loss occurs on rupturing of RBCs as found in diseases namely haemophilia, haemolytic anaemia, diabetes mellitus. TFE can be implied in discovering therapeutic targets, as it mimics the biological membrane environment. A global transition of hemoglobin (Hb) in presence of TFE was studied by using multi-methodological approach. The presence of partially folded state of Hb at 15% v/v TFE was confirmed by altered tryptophan environment, and retention of native-like secondary and tertiary structure. Molten globule state was observed at 20% v/v TFE as detected by increase tryptophan and high ANS fluorescence, slight alterations in Soret band relative to native. TFE on increasing concentration induced protofibrils at 25% v/v and fibrils at 45% v/v as depicted by altered tryptophan environment, heme loss, increase in non-native β-sheet secondary and tertiary structure, large hydrodynamic radii of heme-protein, high ANS, thioflavin T fluorescence and shift in Congo Red absorbance. Comet assay showed that protofibrils are cytotoxic to lymphocytes. SEM and XRD confirmed these aggregates to be fibrillar in nature.

Consequential secondary structure alterations and aggregation during prolonged casein glycation

Non-enzymatic glycosylation (glycation) of casein is a process used not just to ameliorate the quality of dairy products but also to increase the shelf life of canned foods, including baby milk supplements. Incubation of κ-casein with reducing sugars for 15 days at physiological temperature showed the formation of a molten globule state at day 9 and 12 during fructation and glucation respectively. This state exhibits substantial secondary structure and maximum ANS binding. Later on, glycation resulted in the formation of aggregates at day 12 in presence of fructose and day 15 in presence of glucose. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and FTIR. These aggregates showed altered tryptophan environment, decrease ANS binding relative to molten globule state and increase in Thioflavin T fluorescence. Aggregates were also accompanied by the accumulation of AGEs, indicative of structural damage to the protein and formation of potentially harmful species at the physiological level. Fructose was more reactive than glucose and thus caused early and significant changes in the protein. From our studies, we conclude that controlling the extent of the Maillard reaction in the food industry is essential to counter its negative effects and expand its safety spectrum.

Deciphering Structural Intermediates and Genotoxic Fibrillar Aggregates of Albumins: A Molecular Mechanism Underlying for Degenerative Diseases

The misfolding and aggregation of proteins is involved in some of the most prevalent neurodegenerative disorders. The importance of human serum albumin (HSA) stems from the fact that it is involved in bio-regulatory and transport phenomena. Here the effect of acetonitrile (ACN) on the conformational stability of HSA and by comparison, ovalbumin (OVA) has been evaluated in the presence and absence of NaCl. The results show the presence of significant amount of secondary structure in HSA at 70% ACN and in OVA at 50% ACN, as evident from far-UV Circular Dichroism (CD) and Attenuated Total Reflection Fourier transformed infra red spectroscopy (ATR-FTIR). Tryptophan and 8-Anilino-1-Naphthalene-Sulphonic acid (ANS) fluorescence indicate altered tryptophan environment and high ANS binding suggesting a compact “molten globule”-like conformation with enhanced exposure of hydrophobic surface area. However, in presence of NaCl no intermediate state was observed. Detection of aggregates in HSA and OVA was possible at 90% ACN. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and ATR-FTIR. These aggregates exhibit increase Thioflavin T (Th T) fluorescence with a red shift of Congo red (CR) absorption spectrum. X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis confirmed the presence of fibrillar aggregates. Single cell gel electrophoresis (SCGE) assay of these fibrillar aggregates showed the DNA damage resulting in cell necrosis confirming their genotoxic nature. Some proteins not related to any human disease form fibrils in vitro. In the present study ACN gives access to a model system to study the process of aggregation.

Equilibrium studies of cellulase aggregates in presence of ascorbic and boric acid

The aggregate formation of cellulase was detected at 300 and 10mM ascorbic and boric acid respectively. These aggregates showed reduced enzyme activity, loss in near-UV signal, decrease tryptophan and ANS fluorescence. They possess increase in non-native β-sheet structure as evident from far-UV CD and FTIR spectra, large hydrodynamic radii, increase thioflavin T fluorescence and shift in Congo red. Cellulase at 90mM ascorbic acid exists as molten globule with retention of secondary structure, altered tryptophan environment, high ANS binding and loss in tertiary structure. Ascorbic acid acts as an antioxidant up to 90mM and beyond this as a pro-oxidant.

Differing structural characteristics of molten globule intermediate of peanut lectin in urea and guanidine-HCl

The structural characteristics of exclusive equilibrium molten globule-like intermediate formed during peanut lectin unfolding in urea and guanidine hydrochloride (GdnHCl) have been investigated by size-exclusion chromatography, circular dichroism, fluorescence, phosphorescence, and chemical modification. The elution behavior and 8-anilino-1-naphthalenesulfonate binding indicate a less compact tertiary structure in urea than in GdnHCl. Further, the urea-induced intermediate reveals perturbed, nonnative typical β-sheet conformation in contrast to native-like atypical β-structure in GdnHCl. N-bromosuccinimide oxidation shows that none of three tryptophan residues is modified for GdnHCl-induced intermediate while one gets oxidized in urea. Such difference in tryptophan environment is supported by acrylamide quenching (Stern–Volmer constant being 3.2 and 5.8M−1 respectively), and phosphorescence studies at 77K which show a blue-shift of (0, 0) band from 412.4nm (GdnHCl) to 411.4nm (urea). These results may provide important insight into the differential effects of GdnHCl and urea on the structural characteristics of intermediate state(s) in protein folding.

Glycation promotes the formation of genotoxic aggregates in glucose oxidase

This study investigates the effect of pentose sugars (ribose and arabinose) on the structural and chemical modifications in glucose oxidase (GOD) as well as genotoxic potential of this modified form. An intermediate state of GOD was observed on day 12 of incubation having CD minima peaks at 222 and 208nm, characteristic of α-helix and a few tertiary contacts with altered tryptophan environment and high ANS binding. All these features indicate the existence of molten globule state of the GOD with ribose and arabinose on day 12. GOD on day 15 of incubation forms β structures as revealed by CD and FTIR which may be due to its aggregation. Furthermore, GOD on day 15 showed a remarkable increase in Thioflavin T fluorescence at 485nm. Comet assay of lymphocytes and plasmid nicking assay in presence of glycated GOD show DNA damage which confirmed the genotoxicity of advance glycated end products. Hence, our study suggests that glycated GOD results in the formation of aggregates and the advanced glycated end products, which are genotoxic in nature.

Partially Folded Glycated State of Human Serum Albumin Tends to Aggregate

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reactions that results in the formation of advanced glycation end products. We studied the impact of incubation with various sugars for 4 weeks on the behaviour of human serum albumin incubation using CD, fluorescence, UV–Vis spectrophotometry and polyacrylamide gel electrophoresis. Three weeks of incubation of human serum albumin with sugars resulted in the formation of an intermediate state with negative CD peaks at 222 and 208 nm characteristic of α-helix. The form also retained tertiary contacts but with altered tryptophan environment and high ANS binding indicative of molten globule state. Further incubation of human serum albumin for 4 weeks resulted in the formation of an intermediate form with negative CD peak at 217 nm, characteristic of β-sheet, decreased ANS fluorescence and increased thioflavin T fluorescence characteristic of an aggregated state. Prolonged exposure of human serum albumin to reducing sugars thus exerts greater deleterious effects on its structure and formation of aggregates.

Localization and Environment of Tryptophans in Different Structural States of Concanavalin A

We have investigated the localization and environment of tryptophan residues in different quaternary and conformational states (tetrameric, dimeric, monomeric and unfolded) of metallized and demetallized concanavalin A (ConA) by selective chemical modification, fluorescence, and phosphorescence. ConA has four tryptophan residues (Trp 40, Trp 88, Trp 109 and Trp 182) per subunit. The pattern of oxidation by N-bromosuccinimide (NBS) shows that NBS modifies, in dimer, only Trp 182 which remains inaccessible in tetramer, two (Trp 88 along with Trp 182) in monomer, all four in unfolded form in presence of EDTA, and three (possibly Trp 40 along with Trp 88 and Trp 182) in unfolded form from native or remetallized ConA. Utilizing wavelength-selective fluorescence approach, we have observed a red edge excitation shift (REES) of 6-8 nm for tetramer and dimer. A more pronounced REES (11 nm) is observed for oxidized monomer compared to REES (3 nm) for unoxidized species. Acrylamide quenching shows the Stern-Volmer constant (K(SV)) for dimer, monomer, unfolded ConA and unfolded apo-ConA being 3.8, 5.2, 12.8, 14.0 M(-1), respectively. Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 406.2 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 413.2 for dimer and 412.6 nm for monomer, while the (0,0) band of the oxidized monomer is red shifted to 414.4 nm. These results may provide important insight into subtlety of organization and environment of tryptophans in the context of folding and structural studies of oligomeric proteins including lectins.

Metastability of Papain and the Molecular Mechanism for its Sequential Acid-Denaturation

Acid unfolding of non-inhibited papain at pH 2 was studied by means of spectroscopic and electrophoresis techniques as well as activity assays. We found a molten globule like species (A state) similar to that previously reported for bromelain and S-carboxy-methyl-papain. We demonstrated that this A state is not thermodynamically stable but a metastable conformer which decays into an unfolded conformation in a few hours. The mechanism of acid unfolding to the A state proved to be completely irreversible, with a biphasic time evolution of spectroscopic signals characteristic of the existence of a kinetic intermediate. This latter species showed properties in-between native and A state such as secondary structure, exposition of hydrophobic area and tryptophan environment, but a native like hydrodynamic radius. Native papain seems to unfold at acid pH through at least two kinetic barriers, being its pro-region mandatory to conduct and stabilize its active structure. Computer simulations of acid unfolding, followed by ANS docking, identified three regions of cavity formation induced by acid media which might be used as regions to be fortified by protein engineering in the quest for extreme-resistant proteases or as hot-spots for protease inactivation.

Sub-Structures Formed in the Excited State are Responsible for Tryptophan Residues Fluorescence in β-Lactoglobulin

Origin of tryptophan residues fluorescence in β-lactoglobulin is analyzed. Fluorescence lifetimes and spectra of β-lactoglobulin solution are measured at pH going from 2 to 12 and in 6 M guanidine. Tryptophan residues emit with three lifetimes at all conditions. Two lifetimes (0.4-0.5 ns and 2-4 ns) are in the same range of those measured for tryptophan free in solution. Lifetimes in the denatured states are lower than those measured in the native state. Pre-exponential values are modified with the protein structure. Data are identical to those already obtained for other proteins. Fluorescence lifetimes characterize internal states of the tryptophan residues (Tryptophan sub-structures) independently of the tryptophan environments, the third lifetime results from the interaction that is occurring between the Trp residues and its environment. Pre-exponential values characterize substructures populations. In conclusion, tryptophan mission occurs from substates generated in the excited state. This is in good agreement with the theory we described in recent works.

Is 10-100ps Spectral Relaxation of Trp An Indicator of Local Disorder in Proteins?

We have studied the time-resolved fluorescence of the Trp43 residue of the globular protein GB1 upon acid induced equilibrium unfolding.NMR structural experiments have shown this protein is actually very acid stable above pH3.Nanosecond time-resolved TCSPC data clearly suggest that, in the tryptophan environment, partial unfolding appears at surprisingly high pH values. In fact, GB1 exhibits signs of local lifetime changes for pH values as high as 6.9.Further, femtosecond ultraviolet upconversion data reveal a ∼30 ps component with a negative preexponential amplitude in the longer wavelength portion of the emission spectrum. Such a rise time on the red side of emission is a signature of generalized relaxation (solvent and/or protein) around the excited dipole of Trp. A similar term with a ∼2ps exponential is always found for proteins in aqueous solution, representing bulk water motion. Slower terms (10-100ps) have previously been assigned to unusual water environments, protein dipolar relaxation, or the coupling between them.Most intriguing is the fact that, for somewhat lower pH values where GB1 is locally (but not globally) unfolded to a larger degree, the amplitude of the observed 30ps term becomes larger (more negative).Femtosecond Trp emission spectroscopy may thus provide new snapshots of proteins that are “fully folded” over longer time averaging but still have transiently unstructured regions.

The C. elegans B‐cell lymphoma 2 (Bcl‐2) homolog cell death abnormal 9 (CED‐9) associates with and remodels LIPID membranes

Bcl-2 proteins associate with and remodel mitochondria to regulate apoptosis. While the C. elegans Bcl-2 homolog CED-9 constitutively associates with mitochondria, it is unclear whether or not this association reflects an innate ability of CED-9 to directly remodel mitochondrial membranes. To address this question, we have characterized the effects of recombinantly expressed and purified CED-9 on synthetic lipid vesicles. We found that CED-9 associates with anionic lipid vesicles at neutral pH, and that association can occur independently of the C-terminal transmembrane domain. Membrane association changes the environment of CED-9 tryptophans and results in an apparent increase in α-helical structure. Upon association, CED-9 alters the permeability of membranes resulting in leakage of encapsulated dyes. Furthermore, this membrane remodeling promotes membrane fusion upon protonation of CED-9. Bypass of this protonation trigger can be achieved by mutating two conserved glutamates (E187K/E190K) or removing the N-terminal 67 residues. Together, these in vitro results suggest that CED-9 retains the amphitropic ability of mammalian Bcl-2 proteins to associate with cellular membranes. We therefore discuss the possibility that CED-9 and other Bcl-2 homologs localize at mitochondria to regulate mitochondrial homeostasis by either modulating mitochondrial membrane permeability or fusion.

Frontal UV–visible fluorescence polarization measurement for bovine meat ageing assessment

Among the techniques based on light interactions with biological tissues, fluorescence polarization offers a selective means of characterizing the organization of biological tissues. This paper presents a methodology for investigating the fluorescence polarization of muscle tissues in to obtain structural information, and specifically the structural modifications caused by meat ageing. A theoretical model of fluorescence anisotropy based on geometrical distribution and properties of tryptophan, the major fluorophore in muscle tissues, is proposed. Experimental data are fitted with the model and fitting parameters ( C 1, C 2 and τ) are tracked during meat ageing. Results presented demonstrate how the method is able to show muscle structure modification during ageing. They highlight changes in structural proteins along the main axis of myofibrils and changes in the tryptophan environment resulting from the physicochemical and enzymatic processes at work during ageing.

Pea lectin unfolding reveals a unique molten globule fragment chain

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains -a long β-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a β-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ∼ 20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule β-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular β-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ∼2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.

High expression of IMPACT protein promotes resistance to indoleamine 2,3‐dioxygenase‐induced cell death

Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.