Publications by authors named "Brian F Volkman"

170 Publications

Trisubstituted 1,3,5-Triazines: The First Ligands of the sY12-Binding Pocket on Chemokine CXCL12.

ACS Med Chem Lett 2021 Nov 18;12(11):1773-1782. Epub 2021 Oct 18.

Department of Biochemistry, Program in Chemical Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.

CXCL12, a CXC-type chemokine, binds its receptor CXCR4, and the resulting signaling cascade is essential during development and subsequently in immune function. Pathologically, the CXCL12-CXCR4 signaling axis is involved in many cancers and inflammatory diseases and thus has sparked continued interest in the development of therapeutics. Small molecules targeting CXCR4 have had mixed results in clinical trials. Alternatively, small molecules targeting the chemokine instead of the receptor provide a largely unexplored space for therapeutic development. Here we report that trisubstituted 1,3,5-triazines are competent ligands for the sY12-binding pocket of CXCL12. The initial hit was optimized to be more synthetically tractable. Fifty unique triazines were synthesized, and the structure-activity relationship was probed. Using computational modeling, we suggest key structural interactions that are responsible for ligand-chemokine binding. The lipophilic ligand efficiency was improved, resulting in more soluble, drug-like molecules with chemical handles for future development and structural studies.
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http://dx.doi.org/10.1021/acsmedchemlett.1c00388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8592115PMC
November 2021

The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus.

Cell Mol Life Sci 2021 Nov 29;78(21-22):6963-6978. Epub 2021 Sep 29.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Room 18.5.32., 2200, Copenhagen, Denmark.

The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.
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http://dx.doi.org/10.1007/s00018-021-03930-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8558179PMC
November 2021

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Proc Natl Acad Sci U S A 2021 09;118(38)

Institute for Integrative Genome Biology, University of California, Riverside, CA 92521;

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in , tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.
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http://dx.doi.org/10.1073/pnas.2108281118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8463862PMC
September 2021

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis.

Proteins 2021 Aug 20. Epub 2021 Aug 20.

Genomic Sciences and Precision Medicine Center (GSPMC), Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.
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http://dx.doi.org/10.1002/prot.26219DOI Listing
August 2021

The dimeric form of CXCL12 binds to atypical chemokine receptor 1.

Sci Signal 2021 Aug 17;14(696). Epub 2021 Aug 17.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

The pleiotropic chemokine CXCL12 is involved in diverse physiological and pathophysiological processes, including embryogenesis, hematopoiesis, leukocyte migration, and tumor metastasis. It is known to engage the classical receptor CXCR4 and the atypical receptor ACKR3. Differential receptor engagement can transduce distinct cellular signals and effects as well as alter the amount of free, extracellular chemokine. CXCR4 binds both monomeric and the more commonly found dimeric forms of CXCL12, whereas ACKR3 binds monomeric forms. Here, we found that CXCL12 also bound to the atypical receptor ACKR1 (previously known as Duffy antigen/receptor for chemokines or DARC). In vitro nuclear magnetic resonance spectroscopy and isothermal titration calorimetry revealed that dimeric CXCL12 bound to the extracellular N terminus of ACKR1 with low nanomolar affinity, whereas the binding affinity of monomeric CXCL12 was orders of magnitude lower. In transfected MDCK cells and primary human Duffy-positive erythrocytes, a dimeric, but not a monomeric, construct of CXCL12 efficiently bound to and internalized with ACKR1. This interaction between CXCL12 and ACKR1 provides another layer of regulation of the multiple biological functions of CXCL12. The findings also raise the possibility that ACKR1 can bind other dimeric chemokines, thus potentially further expanding the role of ACKR1 in chemokine retention and presentation.
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http://dx.doi.org/10.1126/scisignal.abc9012DOI Listing
August 2021

Age-associated changes in microRNAs affect the differentiation potential of human mesenchymal stem cells: Novel role of miR-29b-1-5p expression.

Bone 2021 12 14;153:116154. Epub 2021 Aug 14.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29403, United States of America; Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29403, United States of America; Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30912, United States of America; Center for Healthy Aging, Medical College of Georgia, Augusta University, Augusta, GA 30912, United States of America. Electronic address:

Age-associated osteoporosis is widely accepted as involving the disruption of osteogenic stem cell populations and their functioning. Maintenance of the local bone marrow (BM) microenvironment is critical for regulating proliferation and differentiation of the multipotent BM mesenchymal stromal/stem cell (BMSC) population with age. The potential role of microRNAs (miRNAs) in modulating BMSCs and the BM microenvironment has recently gained attention. However, miRNAs expressed in rapidly isolated BMSCs that are naïve to the non-physiologic standard tissue culture conditions and reflect a more accurate in vivo profile have not yet been reported. Here we directly isolated CD271 positive (+) BMSCs within hours from human surgical BM aspirates without culturing and performed microarray analysis to identify the age-associated changes in BMSC miRNA expression. One hundred and two miRNAs showed differential expression with aging. Target prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the up-regulated miRNAs targeting genes in bone development pathways were considerably enriched. Among the differentially up-regulated miRNAs the novel passenger strand miR-29b-1-5p was abundantly expressed as a mature functional miRNA with aging. This suggests a critical arm-switching mechanism regulates the expression of the miR-29b-1-5p/3p pair shifting the normally degraded arm, miR-29b-1-5p, to be the dominantly expressed miRNA of the pair in aging. The normal guide strand miR-29b-1-3p is known to act as a pro-osteogenic miRNA. On the other hand, overexpression of the passenger strand miR-29b-1-5p in culture-expanded CD271+ BMSCs significantly down-regulated the expression of stromal cell-derived factor 1 (CXCL12)/ C-X-C chemokine receptor type 4 (SDF-1(CXCL12)/CXCR4) axis and other osteogenic genes including bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (RUNX2). In contrast, blocking of miR-29b-1-5p function using an antagomir inhibitor up-regulated expression of BMP-2 and RUNX2 genes. Functional assays confirmed that miR-29b-1-5p negatively regulates BMSC osteogenesis in vitro. These novel findings provide evidence of a pathogenic anti-osteogenic role for miR-29b-1-5p and other miRNAs in age-related defects in osteogenesis and bone regeneration.
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http://dx.doi.org/10.1016/j.bone.2021.116154DOI Listing
December 2021

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release.

J Biol Chem 2021 08 17;297(2):100975. Epub 2021 Jul 17.

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USA. Electronic address:

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1-NEDD4L WW3 interaction accounts for most of the AMOT-NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW-PPxY core interaction account for the unusually high affinity of the AMOT PPxY1-NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.
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http://dx.doi.org/10.1016/j.jbc.2021.100975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8368996PMC
August 2021

Metamorphic Protein Folding Encodes Multiple Anti- Mechanisms in XCL1.

Pathogens 2021 Jun 17;10(6). Epub 2021 Jun 17.

Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

species cause serious infections requiring prolonged and sometimes toxic therapy. Antimicrobial proteins, such as chemokines, hold great interest as potential additions to the small number of available antifungal drugs. Metamorphic proteins reversibly switch between multiple different folded structures. XCL1 is a metamorphic, antimicrobial chemokine that interconverts between the conserved chemokine fold (an α-β monomer) and an alternate fold (an all-β dimer). Previous work has shown that human XCL1 kills but has not assessed whether one or both XCL1 folds perform this activity. Here, we use structurally locked engineered XCL1 variants and killing assays, adenylate kinase release assays, and propidium iodide uptake assays to demonstrate that both XCL1 folds kill , but they do so via different mechanisms. Our results suggest that the alternate fold kills via membrane disruption, consistent with previous work, and the chemokine fold does not. XCL1 fold-switching thus provides a mechanism to regulate the XCL1 mode of antifungal killing, which could protect surrounding tissue from damage associated with fungal membrane disruption and could allow XCL1 to overcome candidal resistance by switching folds. This work provides inspiration for the future design of switchable, multifunctional antifungal therapeutics.
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http://dx.doi.org/10.3390/pathogens10060762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235156PMC
June 2021

Targeting the CCR6/CCL20 Axis in Entheseal and Cutaneous Inflammation.

Arthritis Rheumatol 2021 Jun 3. Epub 2021 Jun 3.

University of California, Davis, Sacramento.

Objective: To assess the involvement of the CCR6/CCL20 axis in psoriatic arthritis (PsA) and psoriasis (PsO) and to evaluate its potential as a therapeutic target.

Methods: First, we quantified CCL20 levels in peripheral blood and synovial fluid from PsA patients and examined the presence of CCR6+ cells in synovial and tendon tissue. Utilizing an interleukin-23 minicircle DNA (IL-23 MC) mouse model exhibiting key features of both PsO and PsA, we investigated CCR6 and CCL20 expression as well as the preventive and therapeutic effect of CCL20 blockade. Healthy tendon stromal cells were stimulated in vitro with IL-1β to assess the production of CCL20 by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of conditioned media from stimulated tenocytes in inducing T cell migration was interrogated using a Transwell system.

Results: We observed an up-regulation of both CCR6 and CCL20 in the enthesis of IL-23 MC-treated mice, which was confirmed in human biopsy specimens. Specific targeting of the CCR6/CCL20 axis with a CCL20 locked dimer (CCL20LD) blocked entheseal inflammation, leading to profound reductions in clinical and proinflammatory markers in the joints and skin of IL-23 MC-treated mice. The stromal compartment in the tendon was the main source of CCL20 in this model and, accordingly, in vitro activated human tendon cells were able to produce this chemokine and to induce CCR6+ T cell migration, the latter of which could be blocked by CCL20LD.

Conclusion: Our study highlights the pathogenic role of the CCR6/CCL20 axis in enthesitis and introduces the prospect of a novel therapeutic approach for treating patients with PsO and PsA.
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http://dx.doi.org/10.1002/art.41882DOI Listing
June 2021

Targeted biologic inhibition of both tumor cell-intrinsic and intercellular CLPTM1L/CRR9-mediated chemotherapeutic drug resistance.

NPJ Precis Oncol 2021 Mar 2;5(1):16. Epub 2021 Mar 2.

Essential Biotechnology LLC, Big Bend, WI, United States.

Recurrence of therapy-resistant tumors is a principal problem in solid tumor oncology, particularly in ovarian cancer. Despite common complete responses to first line, platinum-based therapies, most women with ovarian cancer recur, and eventually, nearly all with recurrent disease develop platinum resistance. Likewise, both intrinsic and acquired resistance contribute to the dismal prognosis of pancreatic cancer. Our previous work and that of others has established CLPTM1L (cleft lip and palate transmembrane protein 1-like)/CRR9 (cisplatin resistance related protein 9) as a cytoprotective oncofetal protein that is present on the tumor cell surface. We show that CLPTM1L is broadly overexpressed and accumulated on the plasma membrane of ovarian tumor cells, while weakly or not expressed in normal tissues. High expression of CLPTM1L is associated with poor outcome in ovarian serous adenocarcinoma. Robust re-sensitization of resistant ovarian cancer cells to platinum-based therapy was achieved using human monoclonal biologics inhibiting CLPTM1L in both orthotopic isografts and patient-derived cisplatin resistant xenograft models. Furthermore, we demonstrate that in addition to cell-autonomous cytoprotection by CLPTM1L, extracellular CLPTM1L confers resistance to chemotherapeutic killing in an ectodomain-dependent fashion, and that this intercellular resistance mechanism is inhibited by anti-CLPTM1L biologics. Specifically, exosomal CLPTM1L from cisplatin-resistant ovarian carcinoma cell lines conferred resistance to cisplatin in drug-sensitive parental cell lines. CLPTM1L is present in extracellular vesicle fractions of tumor culture supernatants and in patients' serum with increasing abundance upon chemotherapy treatment. These findings have encouraging implications for the use of anti-CLPTM1L targeted biologics in the treatment of therapy-resistant tumors.
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http://dx.doi.org/10.1038/s41698-021-00152-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925570PMC
March 2021

Investigations on T cell transmigration in a human skin-on-chip (SoC) model.

Lab Chip 2021 04;21(8):1527-1539

Department of Physics and Astronomy, University of Manitoba, 30A Sifton Rd, 301 Allen Bldg, Winnipeg, MB R3T 2N2, Canada. and Department of Biosystems Engineering, University of Manitoba, Winnipeg, MB R3T 2N2, Canada and Department of Dermatology, University of California Davis School of Medicine, Sacramento, CA 95816, USA and Department of Immunology, University of Manitoba, Winnipeg, MB R3E 0T5, Canada.

A microfluidics-based three-dimensional skin-on-chip (SoC) model is developed in this study to enable quantitative studies of transendothelial and transepithelial migration of human T lymphocytes in mimicked skin inflammatory microenvironments and to test new drug candidates. The keys results include 1) CCL20-dependent T cell transmigration is significantly inhibited by an engineered CCL20 locked dimer (CCL20LD), supporting the potential immunotherapeutic use of CCL20LD for treating skin diseases such as psoriasis; 2) transepithelial migration of T cells in response to a CXCL12 gradient mimicking T cell egress from the skin is significantly reduced by a sphingosine-1-phosphate (S1P) background, suggesting the role of S1P for T cell retention in inflamed skin tissues; and 3) T cell transmigration is induced by inflammatory cytokine stimulated epithelial cells in the SoC model. Collectively, the developed SoC model recreates a dynamic multi-cellular micro-environment that enables quantitative studies of T cell transmigration at a single cell level in response to physiological cutaneous inflammatory mediators and potential drugs.
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http://dx.doi.org/10.1039/d0lc01194kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058301PMC
April 2021

Molecular mechanics and dynamic simulations of well-known Kabuki syndrome-associated KDM6A variants reveal putative mechanisms of dysfunction.

Orphanet J Rare Dis 2021 02 5;16(1):66. Epub 2021 Feb 5.

Genomic Sciences and Precision Medicine Center (GSPMC), Medical College of Wisconsin, Milwaukee, WI, USA.

Background: Kabuki syndrome is a genetic disorder that affects several body systems and presents with variations in symptoms and severity. The syndrome is named for a common phenotype of faces resembling stage makeup used in a Japanese traditional theatrical art named kabuki. The most frequent cause of this syndrome is mutations in the H3K4 family of histone methyltransferases while a smaller percentage results from genetic alterations affecting the histone demethylase, KDM6A. Because of the rare presentation of the latter form of the disease, little is known about how missense changes in the KDM6A protein sequence impact protein function.

Results: In this study, we use molecular mechanic and molecular dynamic simulations to enhance the annotation and mechanistic interpretation of the potential impact of eleven KDM6A missense variants found in Kabuki syndrome patients. These variants (N910S, D980V, S1025G, C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W, and R1351Q) are predicted to be pathogenic, likely pathogenic or of uncertain significance by sequence-based analysis. Here, we demonstrate, for the first time, that although Kabuki syndrome missense variants are found outside the functionally critical regions, they could affect overall function by significantly disrupting global and local conformation (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), chemical environment (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), and/or molecular dynamics of the catalytic domain (all variants). In addition, our approaches predict that many mutations, in particular C1153R, could allosterically disrupt the key enzymatic interactions of KDM6A.

Conclusions: Our study demonstrates that the KDM6A Kabuki syndrome variants may impair histone demethylase function through various mechanisms that include altered protein integrity, local environment, molecular interactions and protein dynamics. Molecular dynamics simulations of the wild type and the variants are critical to gain a better understanding of molecular dysfunction. This type of comprehensive structure- and MD-based analyses should help develop improved impact scoring systems to interpret the damaging effects of variants in this protein and other related proteins as well as provide detailed mechanistic insight that is not currently predictable from sequence alone.
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http://dx.doi.org/10.1186/s13023-021-01692-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866879PMC
February 2021

Evolution of fold switching in a metamorphic protein.

Science 2021 01;371(6524):86-90

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.

Metamorphic proteins switch between different folds, defying the protein folding paradigm. It is unclear how fold switching arises during evolution. With ancestral reconstruction and nuclear magnetic resonance, we studied the evolution of the metamorphic human protein XCL1, which has two distinct folds with different functions, making it an unusual member of the chemokine family, whose members generally adopt one conserved fold. XCL1 evolved from an ancestor with the chemokine fold. Evolution of a dimer interface, changes in structural constraints and molecular strain, and alteration of intramolecular protein contacts drove the evolution of metamorphosis. Then, XCL1 likely evolved to preferentially populate the noncanonical fold before reaching its modern-day near-equal population of folds. These discoveries illuminate how one sequence has evolved to encode multiple structures, revealing principles for protein design and engineering.
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http://dx.doi.org/10.1126/science.abd8700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017559PMC
January 2021

Specific binding-induced modulation of the XCL1 metamorphic equilibrium.

Biopolymers 2021 Oct 28;112(10):e23402. Epub 2020 Sep 28.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

The metamorphic protein XCL1 switches between two distinct native structures with different functions in the human immune system. This structural interconversion requires complete rearrangement of all hydrogen bonding networks, yet fold-switching occurs spontaneously and reversibly in solution. One structure occupies the canonical α-β chemokine fold and binds XCL1's cognate G-protein coupled receptor, while the other structure occupies a dimeric, all-β fold that binds glycosaminoglycans and has antimicrobial activity. Both of these functions are important for the biologic role of XCL1 in the immune system, and each structure is approximately equally populated under near-physiologic conditions. Recent work has begun to illuminate XCL1's role in combatting infection and cancer. However, without a way to control XCL1's dynamic structural interconversion, it is difficult to study the role of XCL1 fold-switching in human health and disease. Thus, a molecular tool that can regulate the fractional population of the two XCL1 structures is needed. Here, we find by heparin affinity chromatography and NMR that an engineered XCL1 variant called CC5 can trigger a dose-dependent shift in XCL1's metamorphic equilibrium such that the receptor binding structure is depleted, and the antimicrobial structure is more heavily populated. This shift likely occurs due to formation of XCL1-CC5 heterodimers in which both protomers occupy the β-sheet structure. These findings lay the groundwork for future studies seeking to understand the functional role of XCL1 metamorphosis, as well as studies screening for a drug-like molecule that can therapeutically target XCL1 by tuning its metamorphic equilibrium. Moreover, the proof of concept presented here suggests that protein metamorphosis is druggable, opening numerous avenues for controlling biological function of metamorphic proteins by altering the population of their multiple native states.
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http://dx.doi.org/10.1002/bip.23402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004533PMC
October 2021

The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3.

J Biol Chem 2020 10 11;295(40):13927-13939. Epub 2020 Aug 11.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. Electronic address:

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here, we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (Pro-10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and G-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished nearly 500-fold. Deletion of Pro-10 had little effect on CXCL12 binding to the CXCR4 N terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between WT and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N terminus in a subfamily-specific direction.
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http://dx.doi.org/10.1074/jbc.RA120.014244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535910PMC
October 2020

Natural and engineered chemokine (C-X-C motif) receptor 4 agonists prevent acute respiratory distress syndrome after lung ischemia-reperfusion injury and hemorrhage.

Sci Rep 2020 07 9;10(1):11359. Epub 2020 Jul 9.

Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.

We compared therapeutic properties of natural and engineered chemokine (C-X-C motif) receptor 4 (CXCR4) agonists in a rat acute respiratory distress syndrome (ARDS) model utilizing the PaO/FiO-ratio as a clinically relevant primary outcome criterion. Ventilated rats underwent unilateral lung ischemia from t = 0-70 min plus hemorrhage to a mean arterial blood pressure (MAP) of 30 mmHg from t = 40-70 min, followed by reperfusion/fluid resuscitation until t = 300 min. Natural CXCR4 agonists (CXCL12, ubiquitin) and engineered CXCL12 variants (CXCL12, CXCL2, CXCL12K27A/R41A/R47A, CXCL12 (3-68)) were administered within 5 min of fluid resuscitation. Animals treated with vehicle or CXCL12 (3-68) reached criteria for mild and moderate ARDS between t = 90-120 min and t = 120-180 min, respectively, and remained in moderate ARDS until t = 300 min. Ubiquitin, CXCL12, CXCL12 and CXCL12 prevented ARDS development. Potencies of CXCL12/CXCL12/CXCL12 were higher than the potency of ubiquitin. CXCL12K27A/R41A/R47A was inefficacious. CXCL12 > CXCL12 stabilized MAP and reduced fluid requirements. CXCR4 agonists at doses that preserved lung function reduced histological injury of the post-ischemic lung and reduced mortality from 55 to 9%. Our findings suggest that CXCR4 protein agonists prevent development of ARDS and reduce mortality in a rat model, and that development of new engineered protein therapeutics with improved pharmacological properties for ARDS is possible.
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http://dx.doi.org/10.1038/s41598-020-68425-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347544PMC
July 2020

Chemokine CCL28 Is a Potent Therapeutic Agent for Oropharyngeal Candidiasis.

Antimicrob Agents Chemother 2020 07 22;64(8). Epub 2020 Jul 22.

Department of Pediatrics, Division of Infectious Disease, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has antimicrobial activity. In this study, we examine the killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti- activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of Polyamine protein transporters are not required for CCL28 anti- activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti- therapeutic agent without inflammatory side effects.
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http://dx.doi.org/10.1128/AAC.00210-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526824PMC
July 2020

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells.

Bone Rep 2020 Jun 23;12:100270. Epub 2020 Apr 23.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29403, United States of America.

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.
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http://dx.doi.org/10.1016/j.bonr.2020.100270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210406PMC
June 2020

Switchable Membrane Remodeling and Antifungal Defense by Metamorphic Chemokine XCL1.

ACS Infect Dis 2020 05 15;6(5):1204-1213. Epub 2020 Apr 15.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 United States.

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α-β tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4's α-helices are membrane disruptive, while XCL1's helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a β-sheet dimer and the α-β chemokine structure). Here, we investigate XCL1's antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular "Swiss army knife" that can refold into different structures for distinct context-dependent functions: whereas the α-β chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the β-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1's β-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti- activity , in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense.
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http://dx.doi.org/10.1021/acsinfecdis.0c00011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7258946PMC
May 2020

Structural Features of an Extended C-Terminal Tail Modulate the Function of the Chemokine CCL21.

Biochemistry 2020 04 26;59(13):1338-1350. Epub 2020 Mar 26.

From the Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.

The chemokines CCL21 and CCL19, through binding of their cognate receptor CCR7, orchestrate lymph node homing of dendritic cells and naïve T cells. CCL21 differs from CCL19 via an unstructured 32 residue C-terminal domain. Previously described roles for the CCL21 C-terminus include GAG-binding, spatial localization to lymphatic vessels, and autoinhibitory modulation of CCR7-mediated chemotaxis. While truncation of the C-terminal tail induced chemical shift changes in the folded chemokine domain, the structural basis for its influence on CCL21 function remains largely unexplored. CCL21 concentration-dependent NMR chemical shifts revealed weak, nonphysiological self-association that mimics the truncation of the C-terminal tail. We generated a series of C-terminal truncation variants to dissect the C-terminus influence on CCL21 structure and receptor activation. Using NMR spectroscopy, we found that CCL21 residues 80-90 mediate contacts with the chemokine domain. In cell-based assays for CCR7 and ACKR4 activation, we also found that residues 92-100 reduced CCL21 potency in calcium flux, cAMP inhibition, and β-arrestin recruitment. Taken together, these structure-function studies support a model wherein intramolecular interactions with specific residues of the flexible C-terminus stabilize a less active monomer conformation of the CCL21. We speculate that the autoinhibitory intramolecular contacts between the C-terminal tail and chemokine body are disrupted by GAG binding and/or interactions with the CCR7 receptor to ensure optimal functionality.
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http://dx.doi.org/10.1021/acs.biochem.0c00047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307649PMC
April 2020

Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays.

Biochem Biophys Res Commun 2020 07 19;528(2):368-375. Epub 2020 Feb 19.

Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA. Electronic address:

Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α-AR heteromers, however, is lacking. Here we utilized bimolecular luminescence/fluorescence complementation (BiLC/BiFC) combined with intermolecular bioluminescence resonance energy transfer (BRET) assays in HEK293T cells to evaluate CXCR4:α-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGluR) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGluR-EYFP were nonspecific. BRET for CXCR4:ACKR3 and CXCR4:α-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRET of CXCR4:α-AR heteromers without affecting BRET; stimulation with CXCL12 reduced BRET of CXCR4:α-AR heteromers, but did not affect BRET or BRET for CXCR4:α-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRET of CXCR4:α-AR heteromers and increased BRET of CXCR4:α-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α-AR and mGluR homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGluR was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α-ARs is comparable, provide evidence for conformational changes of the receptor complexes upon agonist binding and support the concept that proto- and oligomeric CXCR4 and α-ARs constitutively form higher-order hetero-oligomeric receptor clusters.
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http://dx.doi.org/10.1016/j.bbrc.2020.02.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937191PMC
July 2020

A negative-feedback loop maintains optimal chemokine concentrations for directional cell migration.

Nat Cell Biol 2020 03 10;22(3):266-273. Epub 2020 Feb 10.

Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA.

Chemoattractant gradients frequently guide migrating cells. To achieve the most directional signal, such gradients should be maintained with concentrations around the dissociation constant (K) of the chemoreceptor. Whether this actually occurs in animals is unknown. Here we investigate whether a moving tissue, the zebrafish posterior lateral line primordium, buffers its attractant in this concentration range to achieve robust migration. We find that the Cxcl12 (also known as Sdf1) attractant gradient ranges from 0 to 12 nM, values similar to the 3.4 nM K of its receptor Cxcr4. When we increase the K of Cxcl12 for Cxcr4, primordium migration is less directional. Furthermore, a negative-feedback loop between Cxcl12 and its clearance receptor Ackr3 (also known as Cxcr7) regulates the Cxcl12 concentrations. Breaking this negative feedback by blocking the phosphorylation of the cytoplasmic tail of Ackr3 also results in less directional primordium migration. Thus, directed migration of the primordium is dependent on a close match between the Cxcl12 concentration and the K of Cxcl12 for Cxcr4, which is maintained by buffering of the chemokine levels. Quantitative modelling confirms the plausibility of this mechanism. We anticipate that buffering of attractant concentration is a general mechanism for ensuring robust cell migration.
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http://dx.doi.org/10.1038/s41556-020-0465-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809593PMC
March 2020

Comparative modeling and docking of chemokine-receptor interactions with Rosetta.

Biochem Biophys Res Commun 2020 07 7;528(2):389-397. Epub 2020 Jan 7.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, United States. Electronic address:

Chemokine receptors are a subset of G protein-coupled receptors defined by the distinct property of binding small protein ligands in the chemokine family. Chemokine receptors recognize their ligands by a mechanism that is distinct from other class A GPCRs that bind peptides or small molecules. For this reason, structural information on other ligand-GPCR interactions are only indirectly relevant to understanding the chemokine receptor interface. Additionally, the experimentally determined structures of chemokine-GPCR complexes represent less than 3% of the known interactions of this complex, multi-ligand/multi-receptor network. To enable predictive modeling of the remaining 97% of interactions, a general in silico protocol was designed to utilize existing chemokine receptor crystal structures, co-crystal structures, and NMR ensembles of chemokines bound to receptor fragments. This protocol was benchmarked on the ability to predict each of the three published co-crystal structures, while being blinded to the target structure. Averaging ensembles selected from the top-ranking models reproduced up to 84% of the intermolecular contacts found in the crystal structure, with the lowest Cα-RMSD of the complex at 3.3 Å. The chemokine receptor N-terminus, unresolved in crystal structures, was included in the modeling and recapitulates contacts with known sulfotyrosine binding pockets seen in structures derived from experimental NMR data. This benchmarking experiment suggests that realistic homology models of chemokine-GPCR complexes can be generated by leveraging current structural data.
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http://dx.doi.org/10.1016/j.bbrc.2019.12.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295663PMC
July 2020

Dynamic control of plant water use using designed ABA receptor agonists.

Science 2019 10;366(6464)

Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA.

Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in reveal a role of the type III receptor for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.
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http://dx.doi.org/10.1126/science.aaw8848DOI Listing
October 2019

Structure-function guided modeling of chemokine-GPCR specificity for the chemokine XCL1 and its receptor XCR1.

Sci Signal 2019 09 3;12(597). Epub 2019 Sep 3.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Chemokines interact with their G protein-coupled receptors (GPCRs) through a two-step, two-site mechanism and, through this interaction, mediate various homeostatic and immune response mechanisms. Upon initial recognition of the chemokine by the receptor, the amino terminus of the chemokine inserts into the orthosteric pocket of the GPCR, causing conformational changes that trigger intracellular signaling. There is considerable structural and functional evidence to suggest that the amino acid composition and length of the chemokine amino terminus is critical for GPCR activation, complementing the size and amino acid composition of the orthosteric pocket. However, very few structures of a native chemokine-receptor complex have been solved. Here, we used a hybrid approach that combines structure-function data with Rosetta modeling to describe key contacts within a chemokine-GPCR interface. We found that the extreme amino-terminal residues of the chemokine XCL1 (Val, Gly, Ser, and Glu) contribute a large fraction of the binding energy to its receptor XCR1, whereas residues near the disulfide bond-forming residue Cys modulate XCR1 activation. Alterations in the XCL1 amino terminus changed XCR1 activation, as determined by assessing inositol triphosphate accumulation, intracellular calcium release, and directed cell migration. Computational analysis of XCL1-XCR1 interactions revealed functional contacts involving Glu of XCL1 and Tyr and Arg of XCR1. Subsequent mutation of Tyr and Arg led to diminished binding and activation of XCR1 by XCL1. These findings demonstrate the utility of a hybrid approach, using biological data and homology modeling, to study chemokine-GPCR interactions.
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http://dx.doi.org/10.1126/scisignal.aat4128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733756PMC
September 2019

What doesn't kill you makes you stranger: Dipeptidyl peptidase-4 (CD26) proteolysis differentially modulates the activity of many peptide hormones and cytokines generating novel cryptic bioactive ligands.

Pharmacol Ther 2019 06 10;198:90-108. Epub 2019 Feb 10.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29403, United States; Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29403, United States; Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30912, United States; Department of Orthopaedic Surgery, Medical College of Georgia, Augusta University, Augusta, GA 30912, United States; Center for Healthy Aging, Medical College of Georgia, Augusta University, Augusta, GA, 30912, United States. Electronic address:

Dipeptidyl peptidase 4 (DPP4) is an exopeptidase found either on cell surfaces where it is highly regulated in terms of its expression and surface availability (CD26) or in a free/circulating soluble constitutively available and intrinsically active form. It is responsible for proteolytic cleavage of many peptide substrates. In this review we discuss the idea that DPP4-cleaved peptides are not necessarily inactivated, but rather can possess either a modified receptor selectivity, modified bioactivity, new antagonistic activity, or even a novel activity relative to the intact parent ligand. We examine in detail five different major DPP4 substrates: glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), peptide tyrosine-tyrosine (PYY), and neuropeptide Y (NPY), and stromal derived factor 1 (SDF-1 aka CXCL12). We note that discussion of the cleaved forms of these five peptides are underrepresented in the research literature, and are both poorly investigated and poorly understood, representing a serious research literature gap. We believe they are understudied and misinterpreted as inactive due to several factors. This includes lack of accurate and specific quantification methods, sample collection techniques that are inherently inaccurate and inappropriate, and a general perception that DPP4 cleavage inactivates its ligand substrates. Increasing evidence points towards many DPP4-cleaved ligands having their own bioactivity. For example, GLP-1 can work through a different receptor than GLP-1R, DPP4-cleaved GIP can function as a GIP receptor antagonist at high doses, and DPP4-cleaved PYY, NPY, and CXCL12 can have different receptor selectivity, or can bind novel, previously unrecognized receptors to their intact ligands, resulting in altered signaling and functionality. We believe that more rigorous research in this area could lead to a better understanding of DPP4's role and the biological importance of the generation of novel cryptic ligands. This will also significantly impact our understanding of the clinical effects and side effects of DPP4-inhibitors as a class of anti-diabetic drugs that potentially have an expanding clinical relevance. This will be specifically relevant in targeting DPP4 substrate ligands involved in a variety of other major clinical acute and chronic injury/disease areas including inflammation, immunology, cardiology, stroke, musculoskeletal disease and injury, as well as cancer biology and tissue maintenance in aging.
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http://dx.doi.org/10.1016/j.pharmthera.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883480PMC
June 2019

Modeling the complete chemokine-receptor interaction.

Methods Cell Biol 2019 1;149:289-314. Epub 2018 Nov 1.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, United States. Electronic address:

Chemokines are soluble, secreted proteins that induce chemotaxis of leukocytes and other cells. Migratory cells can sense the chemokine concentration gradient following chemokine binding and activation of chemokine receptors, a subset of the G protein-coupled receptor (GPCR) superfamily. Chemokine receptor signaling plays a central role in cell migration during inflammatory responses as well as in cancer and other diseases. Given their important role in mediating essential pathologic and physiologic processes, chemokines and their receptors are attractive targets for therapeutic development. A better understanding of the molecular basis of chemokine-GPCR interactions will aid in the understanding of the mechanistic basis for chemokine function in disease-related processes, as well as aid in the design of new therapeutics. High resolution protein structures are critical for determining these mechanisms and investigating the interactions between approximately 50 chemokines and 20 chemokine receptors. Currently, three unique structures of chemokine-GPCR complexes have been determined and have greatly broadened our knowledge of this large protein-protein interaction. While these structures represent only a small fraction of clinically relevant chemokines and receptors, they can be exploited as scaffolds for homology modeling to understand the chemokine-GPCR interactions. This chapter presents a specialized methodology to construct and validate models of chemokine-GPCR complexes using the Rosetta software suite.
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http://dx.doi.org/10.1016/bs.mcb.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790067PMC
April 2019

Solution NMR spectroscopy of GPCRs: Residue-specific labeling strategies with a focus on C-methyl methionine labeling of the atypical chemokine receptor ACKR3.

Methods Cell Biol 2019 15;149:259-288. Epub 2018 Nov 15.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, United States. Electronic address:

The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via CH-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.
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http://dx.doi.org/10.1016/bs.mcb.2018.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941889PMC
April 2019

Biased antagonism of CXCR4 avoids antagonist tolerance.

Sci Signal 2018 10 16;11(552). Epub 2018 Oct 16.

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA.

Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.
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http://dx.doi.org/10.1126/scisignal.aat2214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422681PMC
October 2018
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