Publications by authors named "Steven C Almo"

299 Publications

Structural and Functional Characterization of a Biliverdin-Binding Near-Infrared Fluorescent Protein From the Serpin Superfamily.

J Mol Biol 2021 Nov 16;434(2):167359. Epub 2021 Nov 16.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Medicum, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland; Science Center for Genetics and Life Sciences, Sirius University of Science and Technology, Sochi 354340, Russia. Electronic address:

Biliverdin-binding serpins (BBSs) are proteins that are responsible for coloration in amphibians and fluoresce in the near-infrared (NIR) spectral region. Here we produced the first functional recombinant BBS of the polka-dot treefrog Boana punctata (BpBBS), assembled with its biliverdin (BV) chromophore, and report its biochemical and photochemical characterization. We determined the crystal structure of BpBBS at 2.05 Å resolution, which demonstrated its structural homology to the mammalian protease inhibitor alpha-1-antitrypsin. BV interaction with BpBBS was studied and it was found that the N-terminal polypeptide (residues 19-50) plays a critical role in the BV binding. By comparing BpBBS with the available NIR fluorescent proteins and expressing it in mammalian cells, we demonstrated its potential as a NIR imaging probe. These results provide insight into the non-inhibitory function of serpins, provide a basis for improving their performance in mammalian cells, and suggest possible paths for the development of BBS-based fluorescent probes.
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http://dx.doi.org/10.1016/j.jmb.2021.167359DOI Listing
November 2021

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

J Exp Med 2021 Dec 28;218(12). Epub 2021 Oct 28.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.
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http://dx.doi.org/10.1084/jem.20211112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8558838PMC
December 2021

T-cell receptor-specific immunotherapeutics drive selective in vivo HIV and CMV-specific T-cell expansion in humanized mice.

J Clin Invest 2021 Oct 21. Epub 2021 Oct 21.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, United States of America.

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics which recapitulate antigen-specific T-cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently-tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent anti-viral activities. Intravenous injection of HIV or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens, respectively. Notably, these expanded HIV or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections, respectively, in the humanized mice providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.
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http://dx.doi.org/10.1172/JCI141051DOI Listing
October 2021

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Sci Rep 2021 Sep 28;11(1):19220. Epub 2021 Sep 28.

Cue Biopharma, Cambridge, MA, USA.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.
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http://dx.doi.org/10.1038/s41598-021-98716-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8479091PMC
September 2021

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Nature 2021 Sep 15;597(7877):566-570. Epub 2021 Sep 15.

Department of Chemistry, The Pennsylvania State University, University Park, PA, USA.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N-isopentenyladenosine (msiA) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity. The msiA modification is installed onto isopentenyladenosine (iA) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [FeS] cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C hydrogen of the substrate. MiaB also contains an auxiliary [FeS] cluster, which has been implicated in sulfur transfer to C of iA37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C hydrogen of the substrate but only after C has undergone rehybridization from sp to sp. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.
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http://dx.doi.org/10.1038/s41586-021-03904-6DOI Listing
September 2021

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life.

Biochemistry 2021 07 22;60(26):2116-2129. Epub 2021 Jun 22.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.

Viperin is a member of the radical -adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.
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http://dx.doi.org/10.1021/acs.biochem.0c00958DOI Listing
July 2021

Chemical Synthesis of the Antiviral Nucleotide Analogue ddhCTP.

J Org Chem 2021 07 14;86(13):8843-8850. Epub 2021 Jun 14.

Ferrier Research Institute, Victoria University of Wellington, Wellington 6012, New Zealand.

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.
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http://dx.doi.org/10.1021/acs.joc.1c00761DOI Listing
July 2021

INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication.

Nat Commun 2021 05 12;12(1):2743. Epub 2021 May 12.

Department of Genetics, Albert Einstein College of Medicine, New York City, NY, USA.

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding. INI1-Rpt1 and TAR RNA compete with each other for IN binding with similar IC values. INI1-interaction-defective IN mutant viruses are impaired for incorporation of INI1 into virions and for particle morphogenesis. Computational modeling of IN-CTD/TAR complex indicates that the TAR interface phosphates overlap with negatively charged surface residues of INI1-Rpt1 in three-dimensional space, suggesting that INI1-Rpt1 domain structurally mimics TAR. This possible mimicry between INI1-Rpt1 and TAR explains the mechanism by which INI1/SMARCB1 influences HIV-1 late events and suggests additional strategies to inhibit HIV-1 replication.
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http://dx.doi.org/10.1038/s41467-021-22733-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115288PMC
May 2021

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

mSphere 2021 04 21;6(2). Epub 2021 Apr 21.

Montefiore Medical Center, Bronx, New York, USA.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic. Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.
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http://dx.doi.org/10.1128/mSphere.00224-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546701PMC
April 2021

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective.

bioRxiv 2021 Apr 8. Epub 2021 Apr 8.

Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany.

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

Teaser: We revise Remdesivir's mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP.
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http://dx.doi.org/10.1101/2020.08.06.240325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043451PMC
April 2021

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

Cancer Res 2021 04 22;81(7):1788-1801. Epub 2021 Jan 22.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8 cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137529PMC
April 2021

Treatment of severe COVID-19 with convalescent plasma in Bronx, NYC.

JCI Insight 2021 02 22;6(4). Epub 2021 Feb 22.

Albert Einstein College of Medicine, Bronx, New York, USA.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score-matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.
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http://dx.doi.org/10.1172/jci.insight.142270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934933PMC
February 2021

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

ACS Omega 2021 Jan 21;6(1):85-102. Epub 2020 Dec 21.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, United States.

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
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http://dx.doi.org/10.1021/acsomega.0c03512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771249PMC
January 2021

Understanding the Targeting Mechanisms of Multi-Specific Biologics in Immunotherapy with Multiscale Modeling.

iScience 2020 Dec 20;23(12):101835. Epub 2020 Nov 20.

Department of Systems and Computational Biology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Immunotherapeutics are frequently associated with adverse side effects due to the elicitation of global immune modulation. To lower the risk of these side effects, recombinant DNA technology is employed to enhance the selectivity of cell targeting by genetically fusing different biomolecules, yielding new species referred to as multi-specific biologics. The design of new multi-specific biologics is a central challenge for the realization of new immunotherapies. To understand the molecular determinants responsible for regulating the binding between multi-specific biologics and surface-bound membrane receptors, we developed a multiscale computational framework that integrates various simulation approaches covering different timescales and spatial resolutions. Our model system of multi-specific biologics contains two natural ligands of immune receptors, which are covalently tethered by a peptide linker. Using this method, a number of interesting features of multi-specific biologics were identified. Our study therefore provides an important strategy to design the next-generation biologics for immunotherapy.
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http://dx.doi.org/10.1016/j.isci.2020.101835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710644PMC
December 2020

Treatment of Severe COVID-19 with Convalescent Plasma in the Bronx, NYC.

medRxiv 2020 Dec 4. Epub 2020 Dec 4.

Albert Einstein College of Medicine, Bronx, NY.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as treatment for Coronavirus Disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200mL of CCP with a Spike protein IgG titer ≥1:2,430 (median 1:47,385) within 72 hours of admission to propensity score-matched controls cared for at a medical center in the Bronx, between April 13 to May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients <65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients <65 years, but data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy.
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http://dx.doi.org/10.1101/2020.12.02.20242909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724683PMC
December 2020

Immune responses to SARS-CoV-2 infection in hospitalized pediatric and adult patients.

Sci Transl Med 2020 10 21;12(564). Epub 2020 Sep 21.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Children and youth infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have milder disease than do adults, and even among those with the recently described multisystem inflammatory syndrome, mortality is rare. The reasons for the differences in clinical manifestations are unknown but suggest that age-dependent factors may modulate the antiviral immune response. We compared cytokine, humoral, and cellular immune responses in pediatric (children and youth, age <24 years) ( = 65) and adult ( = 60) patients with coronavirus disease 2019 (COVID-19) at a metropolitan hospital system in New York City. The pediatric patients had a shorter length of stay, decreased requirement for mechanical ventilation, and lower mortality compared to adults. The serum concentrations of interleukin-17A (IL-17A) and interferon-γ (IFN-γ), but not tumor necrosis factor-α (TNF-α) or IL-6, were inversely related to age. Adults mounted a more robust T cell response to the viral spike protein compared to pediatric patients as evidenced by increased expression of CD25 on CD4 T cells and the frequency of IFN-γ CD4 T cells. Moreover, serum neutralizing antibody titers and antibody-dependent cellular phagocytosis were higher in adults compared to pediatric patients with COVID-19. The neutralizing antibody titer correlated positively with age and negatively with IL-17A and IFN-γ serum concentrations. There were no differences in anti-spike protein antibody titers to other human coronaviruses. Together, these findings demonstrate that the poor outcome in hospitalized adults with COVID-19 compared to children may not be attributable to a failure to generate adaptive immune responses.
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http://dx.doi.org/10.1126/scitranslmed.abd5487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658796PMC
October 2020

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts.

medRxiv 2020 Sep 11. Epub 2020 Sep 11.

Division of Endocrinology & Diabetes, Department of Medicine, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY 10461, USA.

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.
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http://dx.doi.org/10.1101/2020.09.10.20192187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491531PMC
September 2020

In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography.

Nat Methods 2020 10 14;17(10):1025-1032. Epub 2020 Sep 14.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8 T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8 T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8 T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8 T-cell populations in vivo, which may serve prognostic and diagnostic roles.
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http://dx.doi.org/10.1038/s41592-020-0934-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541633PMC
October 2020

Redesigning HVEM Interface for Selective Binding to LIGHT, BTLA, and CD160.

Structure 2020 11 13;28(11):1197-1205.e2. Epub 2020 Aug 13.

Department of Systems and Computational Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Electronic address:

Herpes virus entry mediator (HVEM) regulates positive and negative signals for T cell activation through co-signaling pathways. Dysfunction of the HVEM co-signaling network is associated with multiple pathologies related to autoimmunity, infectious disease, and cancer, making the associated molecules biologically and therapeutically attractive targets. HVEM interacts with three ligands from two different superfamilies using two different binding interfaces. The engagement with ligands CD160 and B- and T-lymphocyte attenuator (BTLA), members of immunoglobulin superfamily, is associated with inhibitory signals, whereas inflammatory responses are regulated through the interaction with LIGHT from the TNF superfamily. We computationally redesigned the HVEM recognition interfaces using a residue-specific pharmacophore approach, ProtLID, to achieve switchable-binding specificity. In subsequent cell-based binding assays the new interfaces, designed with only single or double mutations, exhibited selective binding to only one or two out of the three cognate ligands.
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http://dx.doi.org/10.1016/j.str.2020.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642093PMC
November 2020

Narrow-Spectrum Antibiotic Targeting of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis.

Biochemistry 2020 07 5;59(27):2562-2575. Epub 2020 Jul 5.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.

Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical -adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen (), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of growth that exclusively targets MqnE. These data provide the foundation for designing effective therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.
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http://dx.doi.org/10.1021/acs.biochem.0c00070DOI Listing
July 2020

Viperin Reveals Its True Function.

Annu Rev Virol 2020 09 30;7(1):421-446. Epub 2020 Jun 30.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; email:

Most cells respond to viral infections by activating innate immune pathways that lead to the induction of antiviral restriction factors. One such factor, viperin, was discovered almost two decades ago based on its induction during viral infection. Since then, viperin has been shown to possess activity against numerous viruses via multiple proposed mechanisms. Most recently, however, viperin was demonstrated to catalyze the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously unknown ribonucleotide. Incorporation of ddhCTP causes premature termination of RNA synthesis by the RNA-dependent RNA polymerase of some viruses. To date, production of ddhCTP by viperin represents the only activity of viperin that links its enzymatic activity directly to an antiviral mechanism in human cells. This review examines the multiple antiviral mechanisms and biological functions attributed to viperin.
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http://dx.doi.org/10.1146/annurev-virology-011720-095930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191541PMC
September 2020

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY, 10461, USA.

Coronavirus disease 2019 ( ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.06.14.150607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310628PMC
June 2020

A metabolic pathway for bile acid dehydroxylation by the gut microbiome.

Nature 2020 06 17;582(7813):566-570. Epub 2020 Jun 17.

Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA, USA.

The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile, promote hepatocellular carcinoma and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. ). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
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http://dx.doi.org/10.1038/s41586-020-2396-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319900PMC
June 2020

Author Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

Nature 2020 Jul;583(7814):E15

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-020-2322-9DOI Listing
July 2020

Mechanistic dissection of the PD-L1:B7-1 co-inhibitory immune complex.

PLoS One 2020 4;15(6):e0233578. Epub 2020 Jun 4.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

The B7 family represents one of the best-studied subgroups within the Ig superfamily, yet new interactions continue to be discovered. However, this binding promiscuity represents a major challenge for defining the biological contribution of each specific interaction. We developed a strategy for addressing these challenges by combining cell microarray and high-throughput FACS methods to screen for promiscuous binding events, map binding interfaces, and generate functionally selective reagents. Applying this approach to the interactions of mPD-L1 with its receptor mPD-1 and its ligand mB7-1, we identified the binding interface of mB7-1 on mPD-L1 and as a result generated mPD-L1 mutants with binding selectivity for mB7-1 or mPD-1. Next, using a panel of mB7-1 mutants, we mapped the binding sites of mCTLA-4, mCD28 and mPD-L1. Surprisingly, the mPD-L1 binding site mapped to the dimer interface surface of mB7-1, placing it distal from the CTLA-4/CD28 recognition surface. Using two independent approaches, we demonstrated that mPD-L1 and mB7-1 bind in cis, consistent with recent reports from Chaudhri A et al. and Sugiura D et al. We further provide evidence that while CTLA-4 and CD28 do not directly compete with PD-L1 for binding to B7-1, they can disrupt the cis PD-L1:B7-1 complex by reorganizing B7-1 on the cell surface. These observations offer new functional insights into the regulatory mechanisms associated with this group of B7 family proteins and provide new tools to elucidate their function in vitro and in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233578PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272049PMC
August 2020

Structure of a single-chain H2A/H2B dimer.

Acta Crystallogr F Struct Biol Commun 2020 May 28;76(Pt 5):194-198. Epub 2020 Apr 28.

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Chromatin is the complex assembly of nucleic acids and proteins that makes up the physiological form of the eukaryotic genome. The nucleosome is the fundamental repeating unit of chromatin, and is composed of ∼147 bp of DNA wrapped around a histone octamer formed by two copies of each core histone: H2A, H2B, H3 and H4. Prior to nucleosome assembly, and during histone eviction, histones are typically assembled into soluble H2A/H2B dimers and H3/H4 dimers and tetramers. A multitude of factors interact with soluble histone dimers and tetramers, including chaperones, importins, histone-modifying enzymes and chromatin-remodeling enzymes. It is still unclear how many of these proteins recognize soluble histones; therefore, there is a need for new structural tools to study non-nucleosomal histones. Here, a single-chain, tailless Xenopus H2A/H2B dimer was created by directly fusing the C-terminus of H2B to the N-terminus of H2A. It is shown that this construct (termed scH2BH2A) is readily expressed in bacteria and can be purified under non-denaturing conditions. A 1.31 Å resolution crystal structure of scH2BH2A shows that it adopts a conformation that is nearly identical to that of nucleosomal H2A/H2B. This new tool is likely to facilitate future structural studies of many H2A/H2B-interacting proteins.
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http://dx.doi.org/10.1107/S2053230X20004604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193513PMC
May 2020

Structures of FOX-4 Cephamycinase in Complex with Transition-State Analog Inhibitors.

Biomolecules 2020 04 27;10(5). Epub 2020 Apr 27.

Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

Boronic acid transition-state analog inhibitors (BATSIs) are partners with β-lactam antibiotics for the treatment of complex bacterial infections. Herein, microbiological, biochemical, and structural findings on four BATSIs with the FOX-4 cephamycinase, a class C β-lactamase that rapidly hydrolyzes cefoxitin, are revealed. FOX-4 is an extended-spectrum class C cephalosporinase that demonstrates conformational flexibility when complexed with certain ligands. Like other β-lactamases of this class, studies on FOX-4 reveal important insights into structure-activity relationships. We show that SM23, a BATSI, shows both remarkable flexibility and affinity, binding similarly to other β-lactamases, yet retaining an IC value < 0.1 μM. Our analyses open up new opportunities for the design of novel transition-state analogs of class C enzymes.
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http://dx.doi.org/10.3390/biom10050671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277225PMC
April 2020

A Binary Arginine Methylation Switch on Histone H3 Arginine 2 Regulates Its Interaction with WDR5.

Biochemistry 2020 10 31;59(39):3696-3708. Epub 2020 Mar 31.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.

Histone H3 arginine 2 (H3R2) is post-translationally modified in three different states by "writers" of the protein arginine methyltransferase (PRMT) family. H3R2 methylarginine isoforms include PRMT5-catalyzed monomethylation (me1) and symmetric dimethylation (me2s) and PRMT6-catalyzed me1 and asymmetric dimethylation (me2a). WD-40 repeat-containing protein 5 (WDR5) is an epigenetic "reader" protein that interacts with H3R2. Previous studies suggested that H3R2me2s specified a high-affinity interaction with WDR5. However, our prior biological data prompted the hypothesis that WDR5 may also interact with H3R2me1. Here, using highly accurate quantitative binding analysis combined with high-resolution crystal structures of WDR5 in complex with unmodified (me0) and me1/me2s l-arginine amino acids and in complex with the H3R2me1 peptide, we provide a rigorous biochemical study and address long-standing discrepancies of this important biological interaction. Despite modest structural differences at the binding interface, our study supports an interaction model regulated by a binary arginine methylation switch: H3R2me2a prevents interaction with WDR5, whereas H3R2me0, -me1, and -me2s are equally permissive.
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http://dx.doi.org/10.1021/acs.biochem.0c00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529705PMC
October 2020

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens.

Proc Natl Acad Sci U S A 2019 09 3;116(38):19126-19135. Epub 2019 Sep 3.

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611;

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ and preQ also occurs. With the exception of YhhQ, shown to transport preQ and preQ, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen , uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as , salvages preQ from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.
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http://dx.doi.org/10.1073/pnas.1909604116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754566PMC
September 2019

An essential bifunctional enzyme in for itaconate dissimilation and leucine catabolism.

Proc Natl Acad Sci U S A 2019 08 18;116(32):15907-15913. Epub 2019 Jul 18.

Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom;

(Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented ()-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.
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http://dx.doi.org/10.1073/pnas.1906606116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689899PMC
August 2019
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