Protein-induced Changes Research Articles

SARS-CoV-2 Spike Protein Induces Oxidative Stress and Senescence in Mouse and Human Lung.

There is concern that people who had COVID-19 will develop pulmonary fibrosis. Using mouse models, we compared pulmonary inflammation following injection of the spike protein of SARS-CoV-2 (COVID-19) to radiation-induced inflammation to demonstrate similarities between the two models. SARS-CoV-2 (COVID-19) induces inflammatory cytokines and stress responses, which are also common to ionizing irradiation-induced acute pulmonary damage. Cellular senescence, which is a late effect following exposure to SARS-CoV-2 as well as radiation, was investigated. We evaluated the effect of SARS-CoV-2 spike protein compared to ionizing irradiation in K18-hACE2 mouse lung, human lung cell lines, and in freshly explanted human lung. We measured reactive oxygen species, DNA double-strand breaks, stimulation of transforming growth factor-beta pathways, and cellular senescence following exposure to SARS-CoV-2 spike protein, irradiation or SARS-COV-2 and irradiation. We also measured the effects of the antioxidant radiation mitigator MMS350 following irradiation or exposure to SARS-CoV-2. SARS-CoV-2 spike protein induced reactive oxygen species, DNA double-strand breaks, transforming growth factor-β signaling pathways, and senescence, which were exacerbated by prior or subsequent ionizing irradiation. The water-soluble radiation countermeasure, MMS350, reduced spike protein-induced changes. In both the SARS-Co-2 and the irradiation mouse models, similar responses were seen indicating that irradiation or exposure to SARS-CoV-2 virus may lead to similar lung diseases such as pulmonary fibrosis. Combination of irradiation and SARS-CoV-2 may result in a more severe case of pulmonary fibrosis. Cellular senescence may explain some of the late effects of exposure to SARS-CoV-2 spike protein and to ionizing irradiation.

Bt protein hasten entomopathogenic fungi-induced death of nontarget pest whitefly by suppressing protective symbionts

The risk assessment of Bacillus thuringiensis (Bt) crops on nontarget pests has received much attention. Despite the knowledge of various beneficial bacterial symbionts in pests, whether Bt proteins affect these symbionts and subsequently alter the pest's ecology remains largely unknown. The whitefly Bemisia tabaci is one of the most serious nontarget pests in Bt cotton. Here, we explored the Bt Cry1Ac protein-induced changes in whitefly symbiont abundance and the subsequent effects on whitefly response against a naturally prevalent entomopathogenic fungus Cordyceps javanica. The obligate symbiont ‘Candidatus Portiera aleyrodidarum’ (hereafter P. aleyrodidarum) as well as facultative symbionts ‘Candidatus Hamiltonella defensa’ (hereafter H. defensa), ‘Candidatus Cardinium hertigii’ (hereafter C. hertigii) and ‘Candidatus Rickettsia bellii’ (hereafter R. bellii) dominate the microbial community of whiteflies. The Bt exposure had no effects on H. defensa infected (H) and H. defensa-C. hertigii doubly infected (HC) whiteflies, but decreased the total copy number of symbionts as well as the R. bellii proportion in H. defensa-C. hertigii- R. bellii triply infected whiteflies (HCR). C. javanica caused whitefly adults 100 % mortality within 8 days. Without Bt protein exposure, HCR whiteflies survived significantly longer than H and HC whiteflies sprayed by C. javanica, suggesting that R. bellii confers protection. However, in Bt-exposed groups, C. javanica generated synchronous death of H, HC and HCR whiteflies. Specifically, in H and HC whiteflies, Bt protein-exposure showed no significant difference in progress of death caused by C. javanica. But in HCR whiteflies, Bt exposure hastened death induced by C. javanica, suppressing the R. bellii-conferred protection. This is the first report revealing that Bt protein altered symbiont community conferred adverse effects on nontarget pests, providing a new perspective for Bt risk assessment and biocontrol strategies of nontarget pests.

Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells.

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1β, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.

Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles.

In-depth understanding of the intricate interactions between biomolecules and nanoparticles is hampered by a lack of analytical methods providing quantitative information about binding kinetics. Herein, we demonstrate how label-free evanescent light-scattering microscopy can be used to temporally resolve specific protein binding to individual surface-bound (∼100 nm) lipid vesicles. A theoretical model is proposed that translates protein-induced changes in light-scattering intensity into bound mass. Since the analysis is centered on individual lipid vesicles, the signal from nonspecific protein binding to the surrounding surface is completely avoided, offering a key advantage over conventional surface-based techniques. Further, by averaging the intensities from less than 2000 lipid vesicles, the sensitivity is shown to increase by orders of magnitude. Taken together, these features provide a new avenue in studies of protein-nanoparticle interaction, in general, and specifically in the context of nanoparticles in medical diagnostics and drug delivery.

Experimental data using candesartan and captopril indicate no double-edged sword effect in COVID-19.

The key link between renin–angiotensin system (RAS) and COVID-19 is ACE2 (angiotensin-converting enzyme 2), which acts as a double-edged sword, because ACE2 increases the tissue anti-inflammatory response but it is also the entry receptor for the virus. There is an important controversy on several drugs that regulate RAS activity and possibly ACE2, and are widely used, particularly by patients most vulnerable to severe COVID-19. In the lung of healthy rats, we observed that candesartan (an angiotensin type-1, AT1, receptor blocker; ARB) and captopril (an ACE inhibitor; ACEI) up-regulated expression of tissue ACE2 and RAS anti-inflammatory axis receptors (AT2 and Mas receptors). This effect was particularly pronounced in rats with metabolic syndrome (obesity, increased blood pressure and hyperglycemia) and aged rats. Treatment of cultures of human type-II pneumocytes with candesartan or captopril induced up-regulation of ACE2 expression in cells. Treatment with viral spike protein induced a decrease in full-length (i.e. transmembrane) ACE2, an increase in levels of a short intracellular ACE2 polypeptide and an increase in ADAM17 activity in cells, together with an increase in levels of soluble ACE2 and major proinflammatory cytokines in the culture medium. Spike protein-induced changes and levels of spike protein internalization in cells were inhibited by pretreatment with the above-mentioned drugs. The results suggest that these drugs increase ACE2 levels and promote the anti-inflammatory RAS axis in the lung. Furthermore, possible up-regulation of viral entry by the drug-induced increase in expression of transmembrane ACE2 is counteracted by additional mechanisms, particularly by drug-induced inhibition of ADAM17 activity.

Probing RNA-Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation.

Recent advances in multi-wavelength analytical ultracentrifugation (MWL-AUC) combine the power of an exquisitely sensitive hydrodynamic-based separation technique with the added dimension of spectral separation. This added dimension has opened up new doors to much improved characterization of multiple, interacting species in solution. When applied to structural investigations of RNA, MWL-AUC can precisely report on the hydrodynamic radius and the overall shape of an RNA molecule by enabling precise measurements of its sedimentation and diffusion coefficients and identify the stoichiometry of interacting components based on spectral decomposition. Information provided in this chapter will allow an investigator to design experiments for probing ion and/or protein-induced global conformational changes of an RNA molecule and exploit spectral differences between proteins and RNA to characterize their interactions in a physiological solution environment.

Redox Tuning via Ligand-Induced Geometric Distortions at a YMn3O4 Cubane Model of the Biological Oxygen Evolving Complex.

The function of proteins involved in electron transfer is dependent on cofactors attaining the necessary reduction potentials. We establish a mode of cluster redox tuning through steric pressure on a synthetic model related to Photosystem II. Resembling the cuboidal [CaMn3O4] subsite of the biological oxygen evolving complex (OEC), [Mn4O4] and [YMn3O4] complexes featuring ligands of different basicity and chelating properties were characterized by cyclic voltammetry. In the absence of ligand-induced distortions, increasing the basicity of the ligands results in a decrease of cluster reduction potential. Contraction of Y-oxo/Y–Mn distances by 0.1/0.15 Å enforced by a chelating ligand results in an increase of cluster reduction potential even in the presence of strongly basic donors. Related protein-induced changes in Ca-oxo/Ca–Mn distances may have similar effects in tuning the redox potential of the OEC through entatic states and may explain the cation size dependence on the progression of the S-state cycle.

Search and Subvert: Minimalist Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes from Gram-positive bacteria are secreted virulence factors that aid in downregulating host immunity. These PI-PLCs are minimalist peripheral membrane enzymes with a distorted (βα)8 TIM barrel fold offering a conserved and stable scaffold for the conserved catalytic amino acids while membrane recognition is achieved mostly through variable loops. Decades of experimental and computational research on these enzymes have revealed the subtle interplay between molecular mechanisms of catalysis and membrane binding, leading to a semiquantitative model for how they find, bind, and cleave their respective substrates on host cell membranes. Variations in sequence and structure of their membrane binding sites may correlate with how enzymes from different Gram-positive bacteria search for their particular targets on the membrane. Detailed molecular characterization of protein-lipid interactions have been aided by cutting-edge methods ranging from 31P field-cycling NMR relaxometry to monitor protein-induced changes in phospholipid dynamics to molecular dynamics simulations to elucidate the roles of electrostatic and cation-π interactions in lipid binding to single molecule fluorescence measurements of dynamic interactions between PI-PLCs and vesicles. This toolkit is readily applicable to other peripheral membrane proteins including orthologues in Gram-negative bacteria and more recently discovered eukaryotic minimalist PI-PLCs.

Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes

Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5′ domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure.

SPECTRAL BEHAVIOR OF INDICATOR DYES IN THE MODEL PROTEIN – LIPID SYSTEMS

The protolytic and partition equilibria of the indicator dyes in the model lipid and protein-lipid systems have been analyzed. A methodological approach has been developed allowing the partition coefficients of the protonated and deprotonated dye forms to be derived from the spectrophotometric measurements. The partitioning of the indicator dye bromothymol blue into the model bilayer membranes composed of phosphatidylcholine and cardiolipin (9:1, mol:mol) has been examined. The partition coefficient of the protonated dye species into a lipid phase has been found to be 5 orders of magnitude higher than that of the deprotonated dye form. This effect has been interpreted in terms of the differences in the charge distribution over the protonated and deprotonated dye ions, preventing the hydrophobic dye-lipid interactions in the latter case. The reduction of the bromothymol blue partitioning into lipid bilayer in the presence of hemoglobin has been attributed to the protein-induced changes in the structure and physicochemical characteristics of the interfacial membrane region. In the practical aspect, the obtained findings may prove of significance in the design of hemosome-based blood substitutes and elucidating the role of hemoglobin in the molecular etiology of the amyloid disorders, particularly, Alzheimer's disease.

Commentary: Rehebbilitating Memory.

Commentary: Rehebbilitating Memory.

Membrane remodeling and mechanics: Experiments and simulations of α-Synuclein

We review experimental and simulation approaches that have been used to determine curvature generation and remodeling of lipid bilayers by membrane-bending proteins. Particular emphasis is placed on the complementary approaches used to study α-Synuclein (αSyn), a major protein involved in Parkinson's disease (PD). Recent cellular and biophysical experiments have shown that the protein 1) deforms the native structure of mitochondrial and model membranes; and 2) inhibits vesicular fusion. Today's advanced experimental and computational technology has made it possible to quantify these protein-induced changes in membrane shape and material properties. Collectively, experiments, theory and multi-scale simulation techniques have established the key physical determinants of membrane remodeling and rigidity: protein binding energy, protein partition depth, protein density, and membrane tension. Despite the exciting and significant progress made in recent years in these areas, challenges remain in connecting biophysical insights to the cellular processes that lead to disease. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Candida albicans Shed Msb2 and Host Mucins Affect the Candidacidal Activity of Salivary Hst 5.

Salivary Histatin 5 (Hst 5) is an antimicrobial peptide that exhibits potent antifungal activity towards Candida albicans, the causative agent of oral candidiasis. However, it exhibits limited activity in vivo, largely due to inactivation by salivary components of both host and pathogen origin. Proteins secreted by C. albicans during infection such as secreted aspartyl proteases (Saps) and shed mucin Msb2 can reduce Hst 5 activity; and human salivary mucins, while suggested to protect Hst 5 from proteolytic degradation, can entrap peptides into mucin gels, thereby reducing bioavailability. We show here that Sap6 that is secreted during hyphal growth reduces Hst 5 activity, most likely a result of proteolytic degradation of Hst 5 since this effect is abrogated with heat inactivated Sap 6. We further show that just like C. albicans shedding Msb2, mammalian mucins, fetuin and porcine gut mucin (that is related to salivary mucins), also reduce Hst 5 activity. However, we identify mucin-like protein-induced changes in C. albicans cell morphology and aggregation patterns, suggesting that the effect of such proteins on Hst 5 cannot be interpreted independently of their effect on yeast cells.

Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer.

A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.

Heterodimerization with Its Splice Variant Blocks the Ghrelin Receptor 1a in a Non-signaling Conformation: A STUDY WITH A PURIFIED HETERODIMER ASSEMBLED INTO LIPID DISCS*

Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.

Low Protein Intake Magnifies Detrimental Effects of Ovariectomy and Vitamin D on Bone

Protein-induced changes in bone and calcium homeostasis could potentially be greater in the elderly and in women at risk for osteoporosis. We hypothesize that a low protein intake would magnify the negative changes in bone metabolism seen in vitamin D (vitD) insufficiency and/or estrogen deficiency. The present study was undertaken to better understand how a low protein diet along with vitD insufficiency could affect bone metabolism using a rodent ovariectomized (OVX) model. Rats (n = 60) underwent ovariectomy (OVX) or sham operation. The first 15 days after surgery, all rats were fed a standard rodent diet. Thereafter, rats (n = 10/group) were fed a low protein diet (LP; 2.5 %) or a control diet (NP; 12.5 %) with 100 IU% vitD (+D; cholecalciferol) or without vitD (-D) for 45 days. The groups were as follows: SHAM + NP + D (control); SHAM + LP + D; SHAM + LP - D; OVX + NP + D; OVX + LP + D; OVX + LP - D. Body weight (BW) of control and OVX + NP + D groups increased while those feeding the LP diet, independently of vitD feedings, decreased (p < 0.05). The OVX + LP - D group presented the lowest serum Ca, phosphorus and osteocalcin levels and the highest CTX levels (p < 0.05). At the end of the study, total skeleton bone mineral content, proximal tibia bone mineral density, bone volume and trabecular number levels decreased as follows: SHAM + NP + D (controls) > SHAM + LP + D > OVX + NP + D > SHAM + LP - D > OVX + LP + D > OVX + LP - D (p < 0.05). A low protein diet negatively affected bone mass and magnified the detrimental effects of vitD and/or estrogen deficiencies.

Binding of the N-terminal Region of Coactivator TIF2 to the Intrinsically Disordered AF1 Domain of the Glucocorticoid Receptor Is Accompanied by Conformational Reorganizations

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.

Characterization of the role of the antioxidant proteins metallothioneins 1 and 2 in an animal model of Alzheimer’s disease

Alzheimer's disease (AD) is by far the most commonly diagnosed dementia, and despite multiple efforts, there are still no effective drugs available for its treatment. One strategy that deserves to be pursued is to alter the expression and/or physiological action of endogenous proteins instead of administering exogenous factors. In this study, we intend to characterize the roles of the antioxidant, anti-inflammatory, and heavy-metal binding proteins, metallothionein-1 + 2 (MT1 + 2), in a mouse model of Alzheimer's disease, Tg2576 mice. Contrary to expectations, MT1 + 2-deficiency rescued partially the human amyloid precursor protein-induced changes in mortality and body weight in a gender-dependent manner. On the other hand, amyloid plaque burden was decreased in the cortex and hippocampus in both sexes, while the amyloid cascade, neuroinflammation, and behavior were affected in the absence of MT1 + 2 in a complex manner. These results highlight that the control of the endogenous production and/or action of MT1 + 2 could represent a powerful therapeutic target in AD.

Fermentable Fiber Ameliorates Fermentable Protein-Induced Changes in Microbial Ecology, but Not the Mucosal Response, in the Colon of Piglets1–3

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21–23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.