Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.     

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ_1–42) fibril formation in vitro and suppressed Aβ_1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.     

Dysregulation of mTOR Signaling after Brain Ischemia

In this review, we provide recent data on the role of mTOR kinase in the brain under physiological conditions and after damage, with a particular focus on cerebral ischemia. We cover the upstream and downstream pathways that regulate the activation state of mTOR complexes. Furthermore, we summarize recent advances in our understanding of mTORC1 and mTORC2 status in ischemia–hypoxia at tissue and cellular levels and analyze the existing evidence related to two types of neural cells, namely glia and neurons. Finally, we discuss the potential use of mTORC1 and mTORC2 as therapeutic targets after stroke.     

The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.     

Aversive Learning Deficits and Depressive-Like Behaviors Are Accompanied by an Increase in Oxidative Stress in a Rat Model of Fetal Alcohol Spectrum Disorders: The Protective Effect of Rapamycin

Fetal alcohol spectrum disorders (FASDs) are one of the most common consequences of ethanol exposure during pregnancy. In adulthood, these disorders can be manifested by learning and memory deficits and depressive-like behavior. Ethanol-induced oxidative stress may be one of the factors that induces FASD development. The mammalian target of the Rapamycin (mTOR) signaling pathway that acts via two distinct multiprotein complexes, mTORC1 and mTORC2, can affect oxidative stress. We investigated whether mTOR-dependent or mTOR-independent mechanisms are engaged in this phenomenon. Thus, Rapamycin—a selective inhibitor of mTORC1, Torin-2—a non-selective mTORC1/mTORC2 inhibitor, and FK-506—a drug that impacts oxidative stress in an mTOR-independent manner were used. Behavioral tests were performed in adult (PND60-65) rats using a passive avoidance (PA) task (aversive learning and memory) and forced swimming test (FST) (depressive-like behaviors). In addition, the biochemical parameters of oxidative stress, such as lipid peroxidation (LPO), as well as apurinic/apyrimidinic (AP)-sites were determined in the hippocampus and prefrontal cortex in adult (PND65) rats. The rat FASD model was induced by intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4–9 (an equivalent to the third trimester of human pregnancy). All substances (3 mg/kg) were given 30 min before ethanol. Our results show that neonatal ethanol exposure leads to deficits in context-dependent fear learning and depressive-like behavior in adult rats that were associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Because these effects were completely reversed by Rapamycin, an mTORC1 inhibitor, this outcome suggests its usefulness as a preventive therapy in disorders connected with prenatal ethanol exposure.     

The Anti-TNF-α Antibody Infliximab Inhibits the Expression of Fat-Transporter-Protein FAT/CD36 in a Selective Hepatic-Radiation Mouse Model

Previously, we reported a radiation-induced inflammation triggering fat-accumulation through fatty-acid-translocase/cluster of differentiation protein 36 (FAT/CD36) in rat liver. Furthermore, inhibition of radiation-induced FAT/CD36-expression by anti-tumor necrosis factor-α (anti-TNF-α) (infliximab) was shown in vitro. The current study investigates fat-accumulation in a mouse-model of single-dose liver-irradiation (25-Gray) and the effect of anti-TNF-α-therapy on FAT/CD36 gene-expression. Mice livers were selectively irradiated in vivo in presence or absence of infliximab. Serum- and hepatic-triglycerides, mRNA, and protein were analyzed by colorimetric assays, RT-PCR, Immunofluorescence and Western-Blot, respectively. Sudan-staining was used demonstrating fat-accumulation in tissue. In mice livers, early (1–3 h) induction of TNF-α-expression, a pro-inflammatory cytokine, was observed. It was followed by elevated hepatic-triglyceride level (6–12 h), compared to sham-irradiated controls. In contrast, serum-triglyceride level was decreased at these time points. Similar to triglyceride level in mice livers, Sudan staining of liver cryosections showed a quick (6–12 h) increase of fat-droplets after irradiation. Furthermore, expression of fat-transporter-protein FAT/CD36 was increased at protein level caused by radiation or TNF-α. TNF-α-blockage by anti-TNF-α showed an early inhibition of radiation-induced FAT/CD36 expression in mice livers. Immunohistochemistry showed basolateral and cytoplasmic expression of FAT/CD36 in hepatocytes. Moreover, co-localization of FAT/CD36 was detected with α-smooth muscle actin (α-SMA⁺) cells and F4/80⁺ macrophages. In summary, hepatic-radiation triggers fat-accumulation in mice livers, involving acute-phase-processes. Accordingly, anti-TNF-α-therapy prevented early radiation-induced expression of FAT/CD36 in vivo.     

The mTORC2 Regulator Homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Osteoblast-Lineage Cells

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including β-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR−/− mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR−/− mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.     

Controlling the Heterodimerisation of the Phytosulfokine Receptor 1 (PSKR1) via Island Loop Modulation

Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing a pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between a leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor’s island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of the PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel “druggable” binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.     

Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.     

Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N⁷/N⁹-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC₅₀ values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.     

Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays)

Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-β-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. K_M values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the K_i value determined as 117 μM, while IAA–Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.     

Design and Experimental Evaluation of a Peptide Antagonist against Amyloid β(1–42) Interactions with Calmodulin and Calbindin-D28k

Amyloid β_1–42 (Aβ(1–42)) oligomers have been linked to the pathogenesis of Alzheimer’s disease (AD). Intracellular calcium (Ca²⁺) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aβ neurotoxicity in AD. The Ca²⁺ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aβ(1–42) oligomers and extensively binds internalized Aβ(1–42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aβ (1–42), using a combined strategy based on the experimental results obtained for Aβ(1–42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aβ(1–42) HiLyte^TM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aβ(1–42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aβ(1–42):CaM and of Aβ(1–42):calbindin-D28k complexes.     

Tetraspanin 8-Rictor-Integrin α3 Complex Is Required for Glioma Cell Migration

The malignant glioma remains one of the most aggressive human malignancies with extremely poor prognosis. Glioma cell invasion and migration are the main causes of death. In the current study, we studied the expression and the potential functions of tetraspanin 8 (Tspan8) in malignant gliomas. We found that Tspan8 expression level is high in both malignant glioma tissues and in several human glioma cell lines, where it formed a complex integrin α3 and rictor, the latter is a key component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Disruption of this complex, through siRNA-mediated knockdown of anyone of these three proteins, inhibited U251MG glioma cell migration in vitro. We further showed that Tspan8-rictor association appeared required for mTORC2 activation. Knockdown of Tspan8 by the targeted siRNAs prevented mTOR-rictor (mTORC2) assembly as well as phosphorylation of AKT (Ser-473) and protein kinase C α (PKCα) in U251MG cells. Together, these results demonstrate that over-expressed Tspan8 in malignant glioma forms a complex with rictor and integrin α3 to mediate mTORC2 activation and glioma cell migration. Therefore, targeting Tspan8-rictor-integrin α3 complex may provide a potential therapeutic intervention for malignant glioma.     

Is the Mitochondrial Membrane Potential (∆Ψ) Correctly Assessed? Intracellular and Intramitochondrial Modifications of the ∆Ψ Probe,Rhodamine 123

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.     

AMPK–mTOR Signaling and Cellular Adaptations in Hypoxia

Cellular energy is primarily provided by the oxidative degradation of nutrients coupled with mitochondrial respiration, in which oxygen participates in the mitochondrial electron transport chain to enable electron flow through the chain complex (I–IV), leading to ATP production. Therefore, oxygen supply is an indispensable chapter in intracellular bioenergetics. In mammals, oxygen is delivered by the bloodstream. Accordingly, the decrease in cellular oxygen level (hypoxia) is accompanied by nutrient starvation, thereby integrating hypoxic signaling and nutrient signaling at the cellular level. Importantly, hypoxia profoundly affects cellular metabolism and many relevant physiological reactions induce cellular adaptations of hypoxia-inducible gene expression, metabolism, reactive oxygen species, and autophagy. Here, we introduce the current knowledge of hypoxia signaling with two-well known cellular energy and nutrient sensing pathways, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). Additionally, the molecular crosstalk between hypoxic signaling and AMPK/mTOR pathways in various hypoxic cellular adaptions is discussed.     

Growth by Insertion: The Family of Bacterial DDxP Proteins

We have identified a variety of proteins in species of the Legionella, Aeromonas, Pseudomonas, Vibrio, Nitrosomonas, Nitrosospira, Variovorax, Halomonas, and Rhizobia genera, which feature repetitive modules of different length and composition, invariably ending at the COOH side with Asp–Asp–x–Pro (DDxP) motifs. DDxP proteins range in size from 900 to 6200 aa (amino acids), and contain 1 to 5 different module types, present in one or multiple copies. We hypothesize that DDxP proteins were modeled by the action of specific endonucleases inserting DNA segments into genes encoding DDxP motifs. Target site duplications (TSDs) formed upon repair of staggered ends generated by endonuclease cleavage would explain the DDxP motifs at repeat ends. TSDs acted eventually as targets for the insertion of more modules of the same or different types. Repeat clusters plausibly resulted from amplification of both repeat and flanking TSDs. The proposed growth shown by the insertion model is supported by the identification of homologous proteins lacking repeats in Pseudomonas and Rhizobium. The 85 DDxP repeats identified in this work vary in length, and can be sorted into short (136–215 aa) and long (243–304 aa) types. Conserved Asp–Gly–Asp–Gly–Asp motifs are located 11–19 aa from the terminal DDxP motifs in all repeats, and far upstream in most long repeats.     

Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CL^pro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CL^pro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CL^pro–PF-07321332 and 3CL^pro–α-ketoamide complexes remained stable compared with 3CL^pro–ritonavir and 3CL^pro–lopinavir. Investigating the dynamic behavior of ligand–protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41–Cys145 of 3CL^pro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CL^pro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.     

Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

Lipopolysaccharide (LPS) is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium). Outer membrane proteins (OMPs) from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46) induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa.