Structural basis and analysis of hamster ACE2 binding to different SARS-CoV-2 spike RBDs.

Pet golden hamsters were first identified being infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta variant of concern (VOC) and transmitted the virus back to humans in Hong Kong in January 2022. Here, we studied the binding of two hamster (golden hamster and Chinese hamster) angiotensin-converting enzyme 2 (ACE2) proteins to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants, including alpha, beta, gamma, delta, and four omicron sub-variants (BA.1, BA.2, BA.3, and BA.4/BA.5). We found that the two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2 (hACE2). Furthermore, the similar infectivity to host cells expressing hamster ACE2s and hACE2 was confirmed with the nine pseudotyped SARS-CoV-2 viruses. Additionally, we determined two cryo-electron microscopy (EM) complex structures of golden hamster ACE2 (ghACE2)/delta RBD and ghACE2/omicron BA.3 RBD. The residues Q34 and N82, which exist in many rodent ACE2s, are responsible for the lower binding affinity of ghACE2 compared to hACE2. These findings suggest that all SARS-CoV-2 VOCs may infect hamsters, highlighting the necessity of further surveillance of SARS-CoV-2 in these animals.IMPORTANCESARS-CoV-2 can infect many domestic animals, including hamsters. There is an urgent need to understand the binding mechanism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to hamster receptors. Herein, we showed that two hamster angiotensin-converting enzyme 2s (ACE2s) (golden hamster ACE2 and Chinese hamster ACE2) can bind to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants and that pseudotyped SARS-CoV-2 viruses can infect hamster ACE2-expressing cells. The binding pattern of golden hamster ACE2 to SARS-CoV-2 RBDs is similar to that of Chinese hamster ACE2. The two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2. We solved the cryo-electron microscopy (EM) structures of golden hamster ACE2 in complex with delta RBD and omicron BA.3 RBD and found that residues Q34 and N82 are responsible for the lower binding affinity of ghACE2 compared to hACE2. Our work provides valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Proficiency in homologous recombination repair is prerequisite for activation of G2-checkpoint at low radiation doses.

Pathways of repair of DNA double strand breaks (DSBs) cooperate with DNA damage cell cycle checkpoints to safeguard genomic stability when cells are exposed to ionizing radiation (IR). It is widely accepted that checkpoints facilitate the function of DSB repair pathways. Whether DSB repair proficiency feeds back into checkpoint activation is less well investigated. Here, we study activation of the G2-checkpoint in cells deficient in homologous recombination repair (HRR) after exposure to low IR doses (∼1 Gy) in the G2-phase. We report that in the absence of functional HRR, activation of the G2-checkpoint is severely impaired. This response is specific for HRR, as cells deficient in classical non-homologous end joining (c-NHEJ) develop a similar or stronger G2-checkpoint than wild-type (WT) cells. Inhibition of ATM or ATR leaves largely unaffected residual G2-checkpoint in HRR-deficient cells, suggesting that the G2-checkpoint engagement of ATM/ATR is coupled to HRR. HRR-deficient cells show in G2-phase reduced DSB-end-resection, as compared to WT-cells or c-NHEJ mutants, confirming the reported link between resection and G2-checkpoint activation. Strikingly, at higher IR doses (≥4 Gy) HRR-deficient cells irradiated in G2-phase activate a weak but readily detectable ATM/ATR-dependent G2-checkpoint, whereas HRR-deficient cells irradiated in S-phase develop a stronger G2-checkpoint than WT-cells. We conclude that HRR and the ATM/ATR-dependent G2-checkpoint are closely intertwined in cells exposed to low IR-doses in G2-phase, where HRR dominates; they uncouple as HRR becomes suppressed at higher IR doses. Notably, this coupling is specific for cells irradiated in G2-phase, and cells irradiated in S-phase utilize a different mechanistic setup.

DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasmid DNA homologous to the mitochondrial genome were efficiently repaired (21 % repair in three hours), whereas a DNA-protein crosslink present on DNA that lacked homology to the mitochondrial genome remained unrepaired. Second, DNA-protein crosslinks present on plasmid DNA lacking homology to the mitochondrial genome were repaired when they were co-electroporated into mitochondria with an undamaged, homologous plasmid DNA molecule. Third, no repair was observed when DNA-protein crosslink-containing plasmids were electroporated into mitochondria isolated from cells pre-treated with the Rad51 inhibitor B02. These findings suggest that mitochondria utilize homologous recombination to repair endogenous and xenobiotic-induced DNA-protein crosslinks. Consistent with this interpretation, cisplatin-induced mitochondrial DNA-protein crosslinks accumulated to higher levels in cells pre-treated with B02 than in control cisplatin-treated cells. These results represent the first evidence of how spontaneous and xenobiotic-induced DNA-protein crosslinks are removed from mitochondrial DNA.

Change on apoptosis, autophagy and mitochondria of the Harderian gland in Cricetulus barabensis during age.

Harderian gland (HG) plays an important role in the physiological adaptation to terrestrial life, however, the mechanisms underlying the changes in the structure and function of the HG during aging remain unclear. This study investigated autophagy and apoptosis in the HG of striped dwarf hamsters (Cricetulus barabensis) of different ages (sub-adult, adult and aged groups) in both males and females. The results showed that LC3II/LC3I and puncta of LC3 were significantly higher in adult and aged individuals than sub-adults, whereas P62 decreased with age. Bax/bcl2was the highest in sub-adults of male and female individuals. Caspase3 activity was the highest in sub-adults of male and female individuals, and the citrate synthase activity was highest in sub-adults of females. ATP synthase, citrate synthase, dynamin-related protein 1 and mitochondrial fission factor (Mff) were the highest in sub-adults of females. Peptidylglycine α-amidating monooxygenase were the highest in the aged group, and those of gonadotropin-releasing hormone was the highest in the adult group. LC3II/LC3I, P62, Drp1, Fis, and bax/bcl2 were higher in males than that in females. These results suggest that apoptosis mainly affects growth and development in the HG, whereas autophagy affects aging. The difference of the HG weight and mitochondrial function between sexes is mainly related to the apoptosis.

Expanded Chinese hamster organ and cell line proteomics profiling reveals tissue-specific functionalities.

Chinese hamster ovary (CHO) cells are the predominant production vehicle for biotherapeutics. Quantitative proteomics data were obtained from two CHO cell lines (CHO-S and CHO DG44) and compared with seven Chinese hamster (Cricetulus griseus) tissues (brain, heart, kidney, liver, lung, ovary and spleen) by tandem mass tag (TMT) labeling followed by mass spectrometry, providing a comprehensive hamster tissue and cell line proteomics atlas. Of the 8470 unique proteins identified, high similarity was observed between CHO-S and CHO DG44 and included increases in proteins involved in DNA replication, cell cycle, RNA processing, and chromosome processing. Alternatively, gene ontology and pathway analysis in tissues indicated increased protein intensities related to important tissue functionalities. Proteins enriched in the brain included those involved in acidic amino acid metabolism, Golgi apparatus, and ion and phospholipid transport. The lung showed enrichment in proteins involved in BCAA catabolism, ROS metabolism, vesicle trafficking, and lipid synthesis while the ovary exhibited enrichments in extracellular matrix and adhesion proteins. The heart proteome included vasoconstriction, complement activation, and lipoprotein metabolism enrichments. These detailed comparisons of CHO cell lines and hamster tissues will enhance understanding of the relationship between proteins and tissue function and pinpoint potential pathways of biotechnological relevance for future cell engineering.

The responses of metabolic rate and neuropeptides to food deprivation in striped hamsters (Cricetulus barabensis) with different basal metabolic rate.

High basal metabolic rate (BMR) is related to a powerful metabolic engine even under food shortage, which can lead to high levels of daily energy expenditure and requires more energy for maintenance in small mammals. To test the hypothesis that animals with different BMR levels respond differently to food shortage, we compared the changes in metabolism, morphology, and gene expression in response to food deprivation (FD) in male-striped hamsters (Cricetulus barabensis) with low (L)- or high (H)-BMR levels. After 36 hr of FD, energy expenditure, metabolic rate (MR), mass of body composition, and leptin and agouti-related peptide gene expressions in the white adipose tissues and the hypothalamus, respectively, decreased significantly in hamsters. The energy expenditure of H-BMR hamsters was reduced more than that of L-BMR hamsters after 36 hr of FD. Furthermore, MR was significantly reduced by FD, and that of the H-BMR group decreased more than that of the L-BMR group during the daytime. Therefore, our data suggest that striped hamsters with different BMR display different responses to variations in food availability. During FD, MR in H-BMR hamsters was more flexible than that in L-BMR animals and L-BMR hamsters could not reduce their MR any lower.

Ganglioside Synthesis by Plasma Membrane-Associated Sialyltransferase in Macrophages.

Gangliosides are constituents of the mammalian cell membranes and participate in the inflammatory response. However, little is known about the presence and enzymatic activity of ganglioside sialyltransferases at the cell surface of macrophages, one of the most important immune cells involved in the innate inflammatory process. In the present study, using biochemical and fluorescent microscopy approaches, we found that endogenous ST8Sia-I is present at the plasma membrane (ecto-ST8Sia-I) of murine macrophage RAW264.7 cells. Moreover, ecto-ST8Sia-I can synthetize GD3 ganglioside at the cell surface in lipopolysaccharide (LPS)-stimulated macrophages even when LPS-stimulated macrophages reduced the total ST8Sia-I expression levels. Besides, cotreatment of LPS with an inhibitor of nitric oxide (NO) synthase recovered the ecto-ST8Sia-I expression, suggesting that NO production is involved in the reduction of ST8Sia-I expression. The diminution of ST8Sia-I expression in LPS-stimulated macrophages correlated with a reduction of GD3 and GM1 gangliosides and with an increment of GD1a. Taken together, the data supports the presence and activity of sialyltransferases at the plasma membrane of RAW264.7 cells. The variations of ecto-ST8Sia-I and ganglioside levels in stimulated macrophages constitutes a promissory pathway to further explore the physiological role of this and others ganglioside metabolism-related enzymes at the cell surface during the immune response.

Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex.

Kv4 pore-forming subunits co-assemble with ß-subunits including KChIP2 and DPP6 and the resultant complexes conduct cardiac transient outward K+ current (Ito). Compound NS5806 has been shown to potentate Ito in canine cardiomyocytes; however, its effects on Ito in other species yet to be determined. We found that NS5806 inhibited native Ito in a concentration-dependent manner (0.1~30 µM) in both mouse ventricular cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but potentiated Ito in the canine cardiomyocytes. In HEK293 cells co-transfected with cloned Kv4.3 (or Kv4.2) and ß-subunit KChIP2, NS5806 significantly increased the peak current amplitude and slowed the inactivation. In contrast, NS5806 suppressed the current and accelerated inactivation of the channels when cells were co-transfected with Kv4.3 (or Kv4.2), KChIP2 and another ß-subunit, DPP6-L (long isoform). Western blot analysis showed that DPP6-L was dominantly expressed in both mouse ventricular myocardium and hiPSC-CMs, while it was almost undetectable in canine ventricular myocardium. In addition, low level of DPP6-S expression was found in canine heart, whereas levels of KChIP2 expression were comparable among all three species. siRNA knockdown of DPP6 antagonized the Ito inhibition by NS5806 in hiPSC-CMs. Molecular docking simulation suggested that DPP6-L may associate with KChIP2 subunits. Mutations of putative KChIP2-interacting residues of DPP6-L reversed the inhibitory effect of NS5806 into potentiation of the current. We conclude that a pharmacological modulator can elicit opposite regulatory effects on Kv4 channel complex among different species, depending on the presence of distinct ß-subunits. These findings provide novel insight into the molecular design and regulation of cardiac Ito. Since Ito is a potential therapeutic target for treatment of multiple cardiovascular diseases, our data will facilitate the development of new therapeutic Ito modulators.

Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6ß, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.

Analysis of the Transcriptional Activity of Retinoic Acid-Related Orphan Receptors (RORs) and Inhibition by Inverse Agonists.

Here, we describe several assays to analyze the transcriptional activity of retinoic acid-related orphan receptors (RORs) and the effect of inverse agonists on their activity. One assay measures the effect of an inverse agonist on the transcriptional activation of a luciferase reporter by RORs in a Tet-On cell system. A mammalian two-hybrid assay analyzes the interaction of the ROR ligand binding domain with a coactivator peptide. Two additional assays examine the effect of an inverse agonist on the activation of a luciferase reporter under control of the promoter of the ROR target gene, IL17, and on ROR-mediated activation using a mammalian monohybrid assay.

LION/web: a web-based ontology enrichment tool for lipidomic data analysis.

BACKGROUND: A major challenge for lipidomic analyses is the handling of the large amounts of data and the translation of results to interpret the involvement of lipids in biological systems. RESULTS: We built a new lipid ontology (LION) that associates >50,000 lipid species to biophysical, chemical, and cell biological features. By making use of enrichment algorithms, we used LION to develop a web-based interface (LION/web, www.lipidontology.com) that allows identification of lipid-associated terms in lipidomes. LION/web was validated by analyzing a lipidomic dataset derived from well-characterized sub-cellular fractions of RAW 264.7 macrophages. Comparison of isolated plasma membranes with the microsomal fraction showed a significant enrichment of relevant LION-terms including "plasma membrane", "headgroup with negative charge", "glycerophosphoserines", "above average bilayer thickness", and "below average lateral diffusion". A second validation was performed by analyzing the membrane fluidity of Chinese hamster ovary cells incubated with arachidonic acid. An increase in membrane fluidity was observed both experimentally by using pyrene decanoic acid and by using LION/web, showing significant enrichment of terms associated with high membrane fluidity ("above average", "very high", and "high lateral diffusion" and "below average transition temperature"). CONCLUSIONS: The results demonstrate the functionality of LION/web, which is freely accessible in a platform-independent way.

Modulation of Proton-Gated Channels by Antidepressants.

The chemical structures of some antidepressants are similar to those of recently described amine-containing ligands of acid-sensing ion channels (ASICs). ASICs are expressed in brain neurons and participate in numerous CNS functions. As such, they can be related to antidepressant action or side effects. We therefore studied the actions of a series of antidepressants on recombinant ASIC1a and ASIC2a and on native ASICs in rat brain neurons. Most of the tested compounds prevented steady-state ASIC1a desensitization evoked by conditioning acidification to pH 7.1. Amitriptyline also potentiated ASIC1a responses evoked by pH drops from 7.4 to 6.5. We conclude that amitriptyline has a twofold effect: it shifts activation to less acidic values while also shifting steady-state desensitization to more acidic values. Chlorpromazine, desipramine, amitriptyline, fluoxetine, and atomoxetine potentiated ASIC2a response. Tianeptine caused strong inhibition of ASIC2a. Both potentiation and inhibition of ASIC2a were accompanied by the slowdown of desensitization, suggesting distinct mechanisms of action on activation and desensitization. In experiments on native heteromeric ASICs, tianeptine and amitriptyline demonstrated the same modes of action as on ASIC2a although with reduced potency.

Fenamates inhibit human sodium channel Nav1.7 and Nav1.8.

Fenamates are N-substituted anthranilic acid derivatives, clinically used as nonsteroidal anti-inflammatory drugs (NSAIDs) in fever, pain and inflammation treatments. Previous studies have shown that they are also modulators of diverse ion channels, exhibiting either activation or inhibitory effects. However, the effects of fenamates on sodium channel subtypes are still unknown. In this study, fenamates, including mefenamic acid, flufenamic acid and tolfenamic acid, were examined by whole-cell patch clamp techniques on the sodium channels hNav1.7 and hNav1.8, which are closely associated with pain. The results showed that the mefenamic acid, flufenamic acid, and tolfenamic acid inhibited the peak currents of hNav1.7 and hNav1.8 in CHO cells stably expressing hNav1.7 and hNav1.8. However, much lighter inhibition effects of hNav1.8 were registered in the experimental system. Furthermore, the mefenamic acid, flufenamic acid and tolfenamic acid significantly affected the inactivation processes of hNav1.7 and hNav1.8 with I-V curves left-shifted to hyperpolarized direction. These data indicate that the inhibition effects of Nav1.7 and Nav1.8 by mefenamic acid, flufenamic acid and tolfenamic acid might contribute to their analgesic activity in addition to their inhibition of cyclooxygenase. These findings provide a basis for further studies in the discovery of other potential targets for NSAIDs.

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.

Heteroarylguanidines as Allosteric Modulators of ASIC1a and ASIC3 Channels.

Acid-sensing ion channels (ASICs) are neuronal Na+-selective ion channels that open in response to extracellular acidification. They are involved in pain, fear, learning, and neurodegeneration after ischemic stroke. 2-Guanidine-4-methylquinazoline (GMQ) was recently discovered as the first nonproton activator of ASIC3. GMQ is of interest as a gating modifier and pore blocker of ASICs. It has however a low potency, and exerts opposite effects on ASIC1a and ASIC3. To further explore the molecular mechanisms of GMQ action, we have used the guanidinium moiety of GMQ as a scaffold and tested the effects of different GMQ derivatives on the ASIC pH dependence and maximal current. We report that GMQ derivatives containing quinazoline and quinoline induced, as GMQ, an alkaline shift of the pH dependence of activation in ASIC3 and an acidic shift in ASIC1a. Another group of 2-guanidinopyridines shifted the pH dependence of both ASIC1a and ASIC3 to more acidic values. Several compounds induced an alkaline shift of the pH dependence of ASIC1a/2a and ASIC2a/3 heteromers. Compared to GMQ, guanidinopyridines showed a 20-fold decrease in the IC50 for ASIC1a and ASIC3 current inhibition at pH 5. Strikingly, 2-guanidino-quinolines and -pyridines showed a concentration-dependent biphasic effect that resulted at higher concentrations in ASIC1a and ASIC3 inhibition (IC50 > 100 µM), while causing at lower concentration a potentiation of ASIC1a, but not ASIC3 currents (EC50 ≈ 10 µM). In conclusion, we describe a new family of small molecules as ASIC ligands and identify an ASIC subtype-specific potentiation by a subgroup of these compounds.

A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells.

DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ∼25% repair at 8 h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (∼60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4 kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ∼38 kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Potentiation and Block of ASIC1a by Memantine.

Acid-sensing ion channels (ASICs) are modulated by various classes of ligands, including the recently described hydrophobic monoamines, which inhibit and potentiate ASICs in a subunit-specific manner. In particular, memantine inhibits ASIC1a and potentiates ASIC2a homomers. The aim of the present work was to characterize action mechanism of memantine on recombinant ASIC1a expressed in CHO (Chinese hamster ovary) cells. We have demonstrated that effect of memantine on ASIC1a strongly depends on membrane voltage, conditioning pH value and application protocol. When applied simultaneously with activating acidification at hyperpolarized voltages, memantine caused the strongest inhibition. Surprisingly, application of memantine between ASIC1a activations at zero voltage caused significant potentiation. Analysis of the data suggests that memantine produces two separate effects, voltage-dependent open-channel block and shift of steady-state desensitization curve to more acidic values. Putative binding sites are discussed based on the computer docking of memantine to the acidic pocket and the pore region.

Molecular Chaperone Receptors.

Extracellular heat shock proteins (HSP) play important roles in cell signaling and immunity. Many of these effects are mediated by surface receptors expressed on a wide range of cell types. We have investigated the nature of such proteins by cloning candidate receptors into cells (CHO-K1) with the rare property of being null for HSP binding. Using this approach we have discovered that Hsp70 binds avidly to at least two classes of receptors including: (1) c-type lectin receptors (CLR) and (2) scavenger receptors (SR). However, the structural nature of the receptor-ligand interactions is not clear at this time. Hsp70 can bind to LOX-1 (a member of both the CLR and SR), with the c-type lectin binding domain (CTLD) as well as the SR family members SREC-I and FEEL-1/CLEVER-1/STABILIN-1, which by contrast have arrays of EGF-like repeats in their extracellular domains. In this chapter we will discuss: (1) methods for discovery of HSP receptors, (2) approaches to the study of individual receptors in cells that contain multiple such receptors, and (3) methods for investigating HSP receptor function in vivo.

Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway.

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.

Synthesis and Radioprotective Activity of Mitochondria Targeted Dihydropyridines In Vitro.

The radiation-induced damage to mitochondrial oxidative respiratory chain could lead to generating of superoxide anions (O2-) and secondary reactive oxygen species (ROS), which are the major resources of continuous ROS production after radiation. Scavenging radiation-induced ROS effectively can help mitochondria to maintain their physiological function and relief cells from oxidative stress. Dihydropyridines (DHPs) are biomimetic hydrogen sources that could protect cells against radiation damage. In this study, we designed and synthetized three novel mitochondrial-targeted dihydropyridines (Mito-DHPs) that utilize the mitochondrial membrane potential to enter the organelle and scavenge ROS. MitoTracker confirmed Mito-DHPs accumulation in mitochondria, and the DCFH-DA assay demonstrated effective ROS scavenging activity. In addition, the γ-H2AX and comet assay demonstrated the ability of Mito-DHPs to protect against both radiation and ROS-induced DNA strand breaks. Furthermore, Mito-DHP1 proved to be non-toxic and displayed significant radioprotection activity (p < 0.05) in vitro. Mito-DHPs are therefore promising antioxidants that could penetrate the membrane of mitochondria, scavenge excessive ROS, and protect cells against radiation-induced oxidative damage.