Bayesian network models identify cooperative GPCR:G protein interactions that contribute to G protein coupling.

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work, we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi, and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.

Mass Spectrometry Defines Lysophospholipids as Ligands for Chicken MHCY Class I Molecules.

Chicken (Gallus gallus) MHCY class I molecules are highly polymorphic yet substantially different from polymorphic MHC class I molecules that bind peptide Ags. The binding grooves in MHCY class I molecules are hydrophobic and too narrow to accommodate peptides. An earlier structural study suggested that ligands for MHCY class I might be lipids, but the contents of the groove were not clearly identified. In this study, lysophospholipids have been identified by mass spectrometry as bound in two MHCY class I isoforms that differ substantially in sequence. The two isoforms, YF1*7.1 and YF1*RJF34, differ by 35 aa in the α1 and α2 domains that form the MHC class I ligand binding groove. Lyso-phosphatidylethanolamine (lyso-PE) 18:1 was the dominant lipid identified in YF1*7.1 and YF1*RJF34 expressed as recombinant molecules and renatured with ß2-microglobulin in the presence of a total lipid extract from Escherichia coli. Less frequently detected were lyso-PE 17:1, lyso-PE 16:1, and lysophosphatidylglycerols 17:1 and 16:0. These data provide evidence that lysophospholipids are candidate ligands for MHCY class I molecules. Finding that MHCY class I isoforms differing substantially in sequence bind the same array of lysophospholipids indicates that the amino acid polymorphism that distinguishes MHCY class I molecules is not key in defining ligand specificity. The polymorphic positions lie mostly away from the binding groove and might define specificity in interactions of MHCY class I molecules with receptors that are presently unidentified. MHCY class I molecules are distinctive in bound ligand and in display of polymorphic residues.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Intrinsically Disordered Proteins: Critical Components of the Wetware.

Despite the wealth of knowledge gained about intrinsically disordered proteins (IDPs) since their discovery, there are several aspects that remain unexplored and, hence, poorly understood. A living cell is a complex adaptive system that can be described as a wetware─a metaphor used to describe the cell as a computer comprising both hardware and software and attuned to logic gates─capable of "making" decisions. In this focused Review, we discuss how IDPs, as critical components of the wetware, influence cell-fate decisions by wiring protein interaction networks to keep them minimally frustrated. Because IDPs lie between order and chaos, we explore the possibility that they can be modeled as attractors. Further, we discuss how the conformational dynamics of IDPs manifests itself as conformational noise, which can potentially amplify transcriptional noise to stochastically switch cellular phenotypes. Finally, we explore the potential role of IDPs in prebiotic evolution, in forming proteinaceous membrane-less organelles, in the origin of multicellularity, and in protein conformation-based transgenerational inheritance of acquired characteristics. Together, these ideas provide a new conceptual framework to discern how IDPs may perform critical biological functions despite their lack of structure.

Dose-dependent progression of multiple low-dose streptozotocin-induced diabetes in mice.

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and although both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function, and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, whereas pathways that were upregulated in both doses were more upregulated in the 35 mg/kg-treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD.

Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.

Efficient and Selective Electrochemically Driven Enzyme-Catalyzed Reduction of Carbon Dioxide to Formate using Formate Dehydrogenase and an Artificial Cofactor.

Increasing levels of carbon dioxide in the atmosphere and the growing need for energy necessitate a shift toward reliance on renewable energy sources and the utilization of carbon dioxide. Thus, producing carbonaceous fuel by the electrochemical reduction of carbon dioxide has been very appealing. We have focused on addressing the principal challenges of poor selectivity and poor energy efficiency in the electrochemical reduction of carbon dioxide. We have demonstrated here a viable pathway for the efficient and continuous electrochemical reduction of CO2 to formate using the metal-independent enzyme type of formate dehydrogenase (FDH) derived from C andida boidinii yeast. This type of FDH is attractive because it is commercially produced. In natural metabolic processes, this type of metal-independent FDH oxidizes formate to carbon dioxide using NAD+ as a cofactor. We show that FDH can catalyze the reverse process to generate formate when the natural cofactor NADH is replaced with an artificial cofactor, the methyl viologen radical cation. The methyl viologen radical cation is generated in situ, electrochemically. Our approach relies on the special properties of methyl viologen as a "unidirectional" redox cofactor for the conversion of CO2 to formate. Methyl viologen (in the oxidized form) does not catalyze formate oxidation, while the methyl viologen radical cation is an effective cofactor for the reduction of carbon dioxide. Thus, although the thermodynamic driving force is favorable for the oxidized form of methyl viologen to oxidize formate to carbon dioxide, the kinetic factors are not favorable. Only the reverse reaction of carbon dioxide reduction to formate is kinetically viable with the cofactor, methyl viologen radical cation. Binding free energy calculated from atomistic molecular dynamics (MD) simulations consolidate our understanding of these special binding properties of the methyl viologen radical cation and its ability to facilitate the two-electron reduction of carbon dioxide to formate in metal-independent FDH. By carrying out the reactions in a novel three-compartment cell, we have demonstrated the continuous production of formate at high energy efficiency and yield. This cell configuration uses judiciously selected ion-exchange membranes to separate the reaction compartments to preserve the yields of the methyl viologen radical cation and formate. By the electroregeneration of the methyl viologen radical cation at -0.44 V versus the normal hydrogen electrode, we could produce formate at 20 mV negative to the reversible electrode potential for carbon dioxide reduction to formate. Our results are in sharp contrast to the large overpotentials of -800 to -1000 mV required on metal catalysts, vindicating the selectivity and kinetic facility provided by FDH. Formate yields as high as 97% ± 1% could be realized by avoiding the adventitious reoxidation of the methyl viologen radical cation by molecular oxygen. We anticipate that the insights from the electrochemical studies and the MD simulations to be useful in redesigning the metal-independent FDH and alternate artificial cofactors to achieve even higher rates of conversion.

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BACKGROUND: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. METHODS: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. RESULTS: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. CONCLUSION: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Identifying Functional Hotspot Residues for Biased Ligand Design in G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several ß-arrestin-biased agonists in ß2-adrenergic receptor (ß2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the κ-opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is ß-arrestin-biased in ß2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both ß2AR and κ-opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.

N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and ß-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

How Do Short Chain Nonionic Detergents Destabilize G-Protein-Coupled Receptors?

Stability of detergent-solubilized G-protein-coupled receptors (GPCRs) is crucial for their purification in a biologically relevant state, and it is well-known that short chain detergents such as octylglucoside are more denaturing than long chain detergents such as dodecylmaltoside. However, the molecular basis for this phenomenon is poorly understood. To gain insights into the mechanism of detergent destabilization of GPCRs, we used atomistic molecular dynamics simulations of thermostabilized adenosine receptor (A2AR) mutants embedded in either a lipid bilayer or detergent micelles of alkylmaltosides and alkylglucosides. A2AR mutants in dodecylmaltoside or phospholipid showed low flexibility and good interhelical packing. In contrast, A2AR mutants in either octylglucoside or nonylglucoside showed decreased α-helicity in transmembrane regions, decreased α-helical packing, and the interpenetration of detergent molecules between transmembrane α-helices. This was not observed in octylglucoside containing phospholipid. Cholesteryl hemisuccinate in dodecylmaltoside increased the energetic stability of the receptor by wedging into crevices on the hydrophobic surface of A2AR, increasing packing interactions within the receptor and stiffening the detergent micelle. The data suggest a three-stage process for the initial events in the destabilization of GPCRs by octylglucoside: (i) highly mobile detergent molecules form small micelles around the receptor; (ii) loss of α-helicity and decreased interhelical packing interactions in transmembrane regions are promoted by increased receptor thermal motion; (iii) transient separation of transmembrane helices allowed penetration of detergent molecules into the core of the receptor. The relative hydration of the headgroup and alkyl chain correlates with detergent harshness and suggests new avenues to develop milder versions of octylglucoside for receptor crystallization.

Differences in allosteric communication pipelines in the inactive and active states of a GPCR.

G-protein-coupled receptors (GPCRs) are membrane proteins that allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to proteins in the intracellular (IC) domain. However, the complete pathway of allosteric communication from the EC to the IC domain, including the role of individual amino acids in the pathway is not known. Using the correlation in torsion angle movements calculated from microseconds-long molecular-dynamics simulations, we elucidated the allosteric pathways in three different conformational states of ß2-adrenergic receptor (ß2AR): 1), the inverse-agonist-bound inactive state; 2), the agonist-bound intermediate state; and (3), the agonist- and G-protein-bound fully active state. The inactive state is less dynamic compared with the intermediate and active states, showing dense clusters of allosteric pathways (allosteric pipelines) connecting the EC with the IC domain. The allosteric pipelines from the EC domain to the IC domain are weakened in the intermediate state, thus decoupling the EC domain from the IC domain and making the receptor more dynamic compared with the other states. Also, the orthosteric ligand-binding site becomes the initiator region for allosteric communication in the intermediate state. This finding agrees with the paradigm that the nature of the agonist governs the specific signaling state of the receptor. These results provide an understanding of the mechanism of allosteric communication in class A GPCRs. In addition, our analysis shows that mutations that affect the ligand efficacy, but not the binding affinity, are located in the allosteric pipelines. This clarifies the role of such mutations, which has hitherto been unexplained.

Structure and dynamics of G-protein coupled receptors.

G-protein coupled receptors (GPCRs) are seven helical transmembrane proteins that mediate cell-to-cell communication. They also form the largest superfamily of drug targets. Hence detailed studies of the three dimensional structure and dynamics are critical to understanding the functional role of GPCRs in signal transduction pathways, and for drug design. In this chapter we compare the features of the crystal structures of various biogenic amine receptors, such as ß1 and ß2 adrenergic receptors, dopamine D3 receptor, M2 and M3 muscarinic acetylcholine receptors. This analysis revealed that conserved residues are located facing the inside of the transmembrane domain in these GPCRs improving the efficiency of packing of these structures. The NMR structure of the chemokine receptor CXCR1 without any ligand bound, shows significant dynamics of the transmembrane domain, especially the helical kink angle on the transmembrane helix6. The activation mechanism of the ß2-adrenergic receptor has been studied using multiscale computational methods. The results of these studies showed that the receptor without any ligand bound, samples conformations that resemble some of the structural characteristics of the active state of the receptor. Ligand binding stabilizes some of the conformations already sampled by the apo receptor. This was later observed in the NMR study of the dynamics of human ß2-adrenergic receptor. The dynamic nature of GPCRs leads to a challenge in obtaining purified receptors for biophysical studies. Deriving thermostable mutants of GPCRs has been a successful strategy to reduce the conformational heterogeneity and stabilize the receptors. This has lead to several crystal structures of GPCRs. However, the cause of how these mutations lead to thermostability is not clear. Computational studies are beginning to shed some insight into the possible structural basis for the thermostability. Molecular Dynamics simulations studying the conformational ensemble of thermostable mutants have shown that the stability could arise from both enthalpic and entropic factors. There are regions of high stress in the wild type GPCR that gets relieved upon mutation conferring thermostability.

Methylglyoxal-Derived Nucleoside Adducts Drive Vascular Dysfunction in a RAGE-Dependent Manner.

Diabetic kidney disease (DKD) is a leading cause of death in patients with diabetes. An early precursor to DKD is endothelial cell dysfunction (ECD), which often precedes and exacerbates vascular disease progression. We previously discovered that covalent adducts formed on DNA, RNA, and proteins by the reactive metabolic by-product methylglyoxal (MG) predict DKD risk in patients with type 1 diabetes up to 16 years pre-diagnosis. However, the mechanisms by which MG adducts contribute to vascular disease onset and progression remain unclear. Here, we report that the most predominant MG-induced nucleoside adducts, N2-(1-carboxyethyl)-deoxyguanosine (CEdG) and N2-(1-carboxyethyl)-guanosine (CEG), drive endothelial dysfunction. Following CEdG or CEG exposure, primary human umbilical vein endothelial cells (HUVECs) undergo endothelial dysfunction, resulting in enhanced monocyte adhesion, increased reactive oxygen species production, endothelial permeability, impaired endothelial homeostasis, and exhibit a dysfunctional transcriptomic signature. These effects were discovered to be mediated through the receptor for advanced glycation end products (RAGE), as an inhibitor for intracellular RAGE signaling diminished these dysfunctional phenotypes. Therefore, we found that not only are MG adducts biomarkers for DKD, but that they may also have a role as potential drivers of vascular disease onset and progression and a new therapeutic modality.

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3.

Decoy oligodeoxynucleotides (ODNs) allow targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated a STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines, K601 and K626, in STAT3 as potential ubiquitination sites. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibition or cereblon deletion, alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B cell-selective C-STAT3DPROTAC. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells, and human and mouse lymphoma cells but not by T cells. C-STAT3DPROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, and MYC). Finally, local C-STAT3DPROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression, while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore the feasibility of using a PROTAC strategy for cell-selective, decoy oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.

Leveraging Cancer Phenotypic Plasticity for Novel Treatment Strategies.

Cancer cells, like all other organisms, are adept at switching their phenotype to adjust to the changes in their environment. Thus, phenotypic plasticity is a quantitative trait that confers a fitness advantage to the cancer cell by altering its phenotype to suit environmental circumstances. Until recently, new traits, especially in cancer, were thought to arise due to genetic factors; however, it is now amply evident that such traits could also emerge non-genetically due to phenotypic plasticity. Furthermore, phenotypic plasticity of cancer cells contributes to phenotypic heterogeneity in the population, which is a major impediment in treating the disease. Finally, plasticity also impacts the group behavior of cancer cells, since competition and cooperation among multiple clonal groups within the population and the interactions they have with the tumor microenvironment also contribute to the evolution of drug resistance. Thus, understanding the mechanisms that cancer cells exploit to tailor their phenotypes at a systems level can aid the development of novel cancer therapeutics and treatment strategies. Here, we present our perspective on a team medicine-based approach to gain a deeper understanding of the phenomenon to develop new therapeutic strategies.

LGR4 is essential for maintaining ß-cell homeostasis through suppression of RANK.

Pancreatic ß-cell stress contributes to diabetes progression. This study demonstrates that Leucine-rich repeat-containing G-protein-coupled-receptor-4 (LGR4) is critical for maintaining ß-cell health and is modulated by stressors. In vitro , Lgr4 knockdown decreases proliferation and survival in rodent ß-cells, while overexpression protects against cytokine-induced cell death in rodent and human ß-cells. Mechanistically, LGR4 suppresses Receptor Activator of Nuclear Factor Kappa B (NFκB) (RANK) and its subsequent activation of NFκB to protect ß-cells. ß-cell-specific Lgr4 -conditional knockout (cko) mice exhibit normal glucose homeostasis but increased ß-cell death in both sexes and decreased proliferation only in females. Male Lgr4 cko mice under stress display reduced ß-cell proliferation and a further increase in ß-cell death. Upon aging, both male and female Lgr4 cko mice display impaired ß-cell homeostasis, however, only female mice are glucose intolerant with decreased plasma insulin. We show that LGR4 is required for maintaining ß-cell health under basal and stress-induced conditions, through suppression of RANK. Teaser: LGR4 receptor is critical for maintaining ß-cell health under basal and stressed conditions, through suppression of RANK.

Critical analysis of the successes and failures of homology models of G protein-coupled receptors.

We present a critical assessment of the performance of our homology model refinement method for G protein-coupled receptors (GPCRs), called LITICon that led to top ranking structures in a recent structure prediction assessment GPCRDOCK2010. GPCRs form the largest class of drug targets for which only a few crystal structures are currently available. Therefore, accurate homology models are essential for drug design in these receptors. We submitted five models each for human chemokine CXCR4 (bound to small molecule IT1t and peptide CVX15) and dopamine D3DR (bound to small molecule eticlopride) before the crystal structures were published. Our models in both CXCR4/IT1t and D3/eticlopride assessments were ranked first and second, respectively, by ligand RMSD to the crystal structures. For both receptors, we developed two types of protein models: homology models based on known GPCR crystal structures, and ab initio models based on the prediction method MembStruk. The homology-based models compared better to the crystal structures than the ab initio models. However, a robust refinement procedure for obtaining high accuracy structures is needed. We demonstrate that optimization of the helical tilt, rotation, and translation is vital for GPCR homology model refinement. As a proof of concept, our in-house refinement program LITiCon captured the distinct orientation of TM2 in CXCR4, which differs from that of adrenoreceptors. These findings would be critical for refining GPCR homology models in future.

Dose-dependent progression of multiple low dose streptozotocin-induced diabetes in mice.

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and while both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, while pathways that were upregulated in both doses were more upregulated in the 35 mg/kg treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

Generation of IL-2-Fc-antibody conjugates by click chemistry.

BACKGROUND: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine. METHODS: We now show that antibodies conjugated by click chemistry to interleukin-2 (IL-2)-Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK. RESULTS: An IL-2-Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL-2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL-2-Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL-2-Fc Par, was selected based on its minimal tendency to aggregate. IL-2-Fc-antibody clicked conjugates retained high IL-2 activity and bound target antigens comparable to parent antibodies. An IL-2-Fc-anti-CEA click conjugate showed comparable anti-tumor activity to an anti-CEA-IL-2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ+ /CD8+ and decreases in FoxP3+ /CD4+ T-cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction. CONCLUSION: The production of antibody targeted IL-2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.