Selective Protein Interaction Research Articles

Comprehensive characterization of polyproline tri-helix macrocyclic nanoscaffolds for predictive ligand positioning.

Multivalent ligands hold promise for enhancing avidity and selectivity to simultaneously target multimeric proteins, as well as potentially modulating receptor signaling in pharmaceutical applications. Essential for these manipulations are nanosized scaffolds that precisely control ligand display patterns, which can be achieved by using polyproline oligo-helix macrocyclic nanoscaffolds via selective binding to protein oligomers and cell surface receptors. This work focuses on synthesis and structural characterization of different-sized polyproline tri-helix macrocyclic (PP3M) scaffolds. Through combined analysis of circular dichroism (CD), small- and wide-angle X-ray scattering (SWAXS), electron spin resonance (ESR) spectroscopy, and molecular modeling, a non-coplanar tri-helix loop structure with partially crossover helix ends is elucidated. This structural model aligns well with scanning tunneling microscopy (STM) imaging. The present work enhances the precision of nanoscale organic synthesis, offering prospects for controlled ligand positioning on scaffolds. This advancement paves the way for further applications in nanomedicine through selective protein interaction, manipulation of cell surface receptor functions, and developments of more complex polyproline-based nanostructures.

TRUPATH: An Open-Source Biosensor Platform for Interrogating the GPCR Transducerome.

G protein-coupled receptors (GPCRs) are the most highly targeted protein family by United States Food and Drug Administration-approved drugs. Despite their historic and continued importance as drug targets, their therapeutic potential remains underexplored and underexploited. While it has been known for some time that GPCRs are able to engage multiple signaling pathways, the majority of drug research and development has followed the older dogma of a single primary pathway for each receptor. This has been due in part to historical reasons, or to a lack of appreciation of the potential to exploit specific pathways over others as a therapeutic modality. Additionally, only recently have technologies been developed to discern selective GPCR-G protein interactions. In this chapter, we introduce TRUPATH, a bioluminescence resonance energy transfer (BRET)-based platform that allows the unambiguous measurement of receptor-catalyzed dissociation or rearrangement of 14 Gα subunits from their respective Gβ and Gγ subunits. Specifically, we provide a detailed protocol for TRUPATH plasmid transfection, microplate preparation, assay implementation, and data analysis. In doing so, we create a template for using TRUPATH to answer basic biological questions, such as "To which G proteins does a given GPCR couple?", and facilitate drug discovery efforts to identify ligands with intra- and inter-G protein family pathway selectivity.

Synovial Sarcoma: A Complex Disease with Multifaceted Signaling and Epigenetic Landscapes.

Aside from a characteristic SS18-SSX translocation identified in almost all cases, no genetic anomalies have been reliably isolated yet to drive the pathogenesis of synovial sarcoma. In the following review, we explore the structural units of wild-type SS18 and SSX, particularly as they relate to the transcriptional alterations and cellular pathway changes imposed by SS18-SSX. Native SS18 and SSX contribute recognizable domains to the SS18-SSX chimeric proteins, which inflict transcriptional and epigenetic changes through selective protein interactions involving the SWI/SNF and Polycomb chromatin remodeling complexes. Multiple oncogenic and developmental pathways become altered, collectively reprogramming the cellular origin of synovial sarcoma and promoting its malignant transformation. Synovial sarcoma is characterized by complex epigenetic and signaling landscapes. Identifying the operational pathways and concomitant genetic changes induced by SS18-SSX fusions could help develop tailored therapeutic strategies to ultimately improve disease control and patient survivorship.

KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA's acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.

Antitumor Metallodrugs that Target Proteins.

Anticancer platinum-based drugs are widely used in the treatment of a variety of tumorigenic diseases. They have been identified to target DNA and thereby induce apoptosis in cancer cells. Their reactivity to biomolecules other than DNA has often been associated with side effects that many cancer patients experience during chemotherapy. The development of metal compounds that target proteins rather than DNA has the potential to overcome or at least reduce the disadvantages of commonly used chemotherapeutics. Many exciting new metal complexes with novel modes of action have been reported and their anticancer activity was linked to selective protein interaction that may lead to improved accumulation in the tumor, higher selectivity and/or enhanced antiproliferative efficacy. The development of new lead structures requires bioanalytical methods to confirm the hypothesized modes of action or identify new, previously unexplored biological targets and pathways. We have selected original developments for review in this chapter and highlighted compounds on track toward clinical application.

Medicinal Chemistry of Gold Anticancer Metallodrugs.

Since ancient times gold and its complexes have been used as therapeutics against different diseases. In modern medicine gold drugs have been applied for the treatment of rheumatoid arthritis, however, recently other medical applications have come into the focus of inorganic medicinal chemistry. This chapter provides a non-comprehensive overview of key developments in the field of gold anticancer drugs. Exciting findings on gold(I) and gold(III) complexes as antitumor agents are summarized together with a discussion of relevant aspects of their modes of action.

PFI-1, a Highly Selective Protein Interaction Inhibitor, Targeting BET Bromodomains

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target.

Functionalization of Graphene Oxide Generates a Unique Interface for Selective Serum Protein Interactions

Potential toxicity and risk of inducing allergy and inflammation have always been a great concern of using nanomaterials in biomedicine. In this work, we investigate the serum behaviors of graphene oxide (GO) and how such behaviors are affected by its surface modification such as PEGylation. The results show that, when incubated with human sera, unfunctionalized GO adsorbs a significant amount of serum proteins and strongly induces complement C3 cleavage (part of the complement activation cascade), generating C3a/C3a(des-Arg), an anaphylatoxin involved in local inflammatory responses, whereas PEGylated nano-GO (nGO-PEG) exhibits dramatic reductions in both protein binding in general and complement C3 activation. Moreover, we uncover that PEGylation on GO nanosheets apparently generates an interesting nanointerface, evidenced by the acquired certain selectivity and increased binding capacities of nGO-PEG toward a few serum proteins. Further mass spectrometry analysis identifies six nGO-PEG binding proteins, four of which are immune-related factors, including C3a/C3a(des-Arg). A series of Western blot analysis demonstrate that nGO-PEG binds up to 2-fold amount of C3a/C3a(des-Arg) than unfunctionalized GO, and can efficiently decrease the level of C3a/C3a(des-Arg) in treated sera, preventing the normal interaction of C3a with its receptor. In a proof-of-concept experiment, we demonstrate that nGO-PEG may serve to help eliminate the C3a/C3a(des-Arg) induced by other nanomaterials such as as-made GO, indicating a new strategy to modulate the immune responses evoked by one nanomaterial through the addition of another type of nanomaterial. Our results highlight the great importance of nanobio interface in regulating the biological effects of nanomaterials.

Role of the adaptor protein LNK in normal and malignant hematopoiesis

The signal transduction pathways, orchestrating the differentiation of hematopoietic stem and progenitor cells in response to cytokine stimulation, are strictly controlled by networks of feedback loops, highly selective protein interactions and finely tuned on/off switches. In hematological malignancies, the aberrant activation of signaling pathways is usually associated with mutations in tyrosine kinases. Recently, the role of negative signaling regulators is increasingly being recognized as an alternative mechanism involved in diseases such as leukemias and myeloproliferative neoplasms (MPNs). The adaptor protein LNK (Src homology 2 (SH2)B3) is a negative regulator of cytokine signaling that has an essential, nonredundant role in normal hematopoiesis. Indeed, LNK-deficient mice show marked expansion of early hematopoietic precursors, more mature myeloid and B-lineage lymphoid cells, as well as enhanced hematopoietic reconstitution. Murine models show that loss of LNK enhances the development of MPNs and may have a role in additional pathologies. LNK mutations were recently identified in patients with MPNs, and studies in animal models and hematopoietic cell lines suggest that LNK controls the aberrant signaling pathways induced by activated oncogenic kinases. In addition, genome-wide studies show that LNK is associated with autoimmune and cardiovascular disorders. These findings have implications for the future study of hematopoiesis, as well as for the development of novel stem cell and disease-specific therapies.

Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family

Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure–activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors.

Crosslinking Studies of Protein-Protein Interactions in Nonribosomal Peptide Biosynthesis

Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.

The Structure of Sox17 Bound to DNA Reveals a Conserved Bending Topology but Selective Protein Interaction Platforms

Sox17 regulates endodermal lineage commitment and is thought to function antagonistically to the pluripotency determinant Sox2. To investigate the biochemical basis for the distinct functions of Sox2 and Sox17, we solved the crystal structure of the high mobility group domain of Sox17 bound to a DNA element derived from the Lama1 enhancer using crystals diffracting to 2.7 Å resolution. Sox17 targets the minor groove and bends the DNA by approximately 80°. The DNA architecture closely resembles the one seen for Sox2/DNA structures, suggesting that the degree of bending is conserved between both proteins and nucleotide substitutions have only marginal effects on the bending topology. Accordingly, affinities of Sox2 and Sox17 for the Lama1 element were found to be identical. However, when the Oct1 contact interface of Sox2 is compared with the corresponding region of Sox17, a significantly altered charge distribution is observed, suggesting differential co-factor recruitment that may explain their biological distinctiveness.

Kinetics and functional studies on interaction between the replicase proteins of Tomato Bushy Stunt Virus: Requirement of p33:p92 interaction for replicase assembly

The assembly of the functional replicase complex via protein:protein and RNA:protein interactions among the viral-coded proteins, host factors and the viral RNA on cellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have characterized essential interactions between p33:p33 and p33:p92 replication proteins of Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome. Surface plasmon resonance (SPR) measurements with purified recombinant p33 and p92 demonstrate that p33 interacts with p92 in vitro and that the interaction requires the S1 subdomain, whereas the S2 subdomain plays lesser function. Kinetic SPR analyses showed that binding of S1 subdomain to the C-terminal half of p33 takes place with moderate binding affinity in the nanomolar range whereas S2 subdomain binds to p33 with micromolar affinity. Using mutated p33 and p92 proteins, we identified critical amino acid residues within the p33:p92 interaction domain that play essential role in replication and the assembly of the tombusviral replicase. In addition, we show that interaction takes place between replication proteins of TBSV and the closely related Cucumber necrosis virus but not between TBSV and the more distantly related Turnip crinkle virus, suggesting that selective protein interactions might prevent the assembly of chimeric replicases carrying replication proteins from different viruses during mixed infections.

Key Molecular Contacts Promote Recognition of the BAFF Receptor by TNF Receptor-Associated Factor 3: Implications for Intracellular Signaling Regulation

B cell-activating factor belonging to the TNF family receptor (BAFF-R), a member of the TNFR superfamily, plays a role in autoimmunity after ligation with BAFF ligand (also called TALL-1, BLyS, THANK, or zTNF4). BAFF/BAFF-R interactions are critical for B cell regulation, and signaling from this ligand-receptor complex results in NF-kappaB activation. Most TNFRs transmit signals intracellularly by recruitment of adaptor proteins called TNFR-associated factors (TRAFs). However, BAFF-R binds only one TRAF adaptor, TRAF3, and this interaction negatively regulates activation of NF-kappaB. In this study, we report the crystal structure of a 24-residue fragment of the cytoplasmic portion of BAFF-R bound in complex with TRAF3. The recognition motif (162)PVPAT(166) in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTbetaR, but is presented in a completely different structural framework. This region of BAFF-R assumes an open conformation with two extended strands opposed at right angles that each make contacts with TRAF3. The recognition motif is located in the N-terminal arm and intermolecular contacts mediate TRAF recognition. In the C-terminal arm, key stabilizing contacts are made, including critical hydrogen bonds with Gln(379) in TRAF3 that define the molecular basis for selective binding of BAFF-R solely to this member of the TRAF family. A dynamic conformational adjustment of Tyr(377) in TRAF3 occurs forming a new intermolecular contact with BAFF-R that stabilizes the complex. The structure of the complex provides a molecular explanation for binding affinities and selective protein interactions in TNFR-TRAF interactions.

Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the data provided in this study indicate that PcTS/PcTS-Cu(2+) could be considered as possible candidates for the development of therapeutic or prophylactic strategies against the alpha-synuclein-related neurodegenerative disorders.

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.

Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol)

Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces.

The biologic activity of polyanions: Past history and new prospectives

AbstractThe toxicology of synthetic polyanions relates to molecular weight. This developed from experience with pyran copolymer (divinyl ether maleic anhydride copolymer), which was tested clinically as an antitumor agent and found to be too toxic for continued clinical trial. Synthetic polyanions of low molecular weight are potent stimuli to macrophage phagocytic function and possess antitumor activity. Increased host resistance to pathogens including bacteria, fungi, viruses, and trypanosomes is seen. In contrast to lower‐molecular‐weight fractions, higher‐molecular‐weight samples inhibit macrophage function, and apparently increased molecular weight &gt; 15,000 is necessary for antiviral and immunologic stimulating capacity. One possible mechanism for the action of polyanions may relate to redistribution of calcium ions within the cell and increasing cyclic GMP activity. This is a redefinition for the role of polyanions in sol–gel alteration in cytoplasm. The killing effect of the polyanion‐activated macrophage on tumor cells resembles that of the polyanion on the nucleus. Polyanions may be potent controllers of the stability of DNA within the nucleus and can act to derepress cells to turn on protein synthesis or, alternatively, inhibit enzymes involved in virus synthesis and control of RNA or DNA polymerase. Thus, they are cogent tools in an understanding of the physiology of nuclear interaction. Biological use for these agents can be developed not only in clinical approaches to inhibition of tumor or viral infection, but to eliminate plutonium, and provide selective enzyme inhibition and protein interaction which allows for isolation, neutralization, and characterization of blood and tissue protein and glycoprotein fractions vital for a wide range of pathophysiology.