ABSTRACT
Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.
ABSTRACT
Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O2 â-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pKa due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.
ABSTRACT
Proliferating cell nuclear antigen (PCNA), the homotrimeric eukaryotic sliding clamp protein, recruits and coordinates the activities of a multitude of proteins that function on DNA at the replication fork. Chromatin assembly factor 1 (CAF-1), one such protein, is a histone chaperone that deposits histone proteins onto DNA immediately following replication. The interaction between CAF-1 and PCNA is essential for proper nucleosome assembly at silenced genomic regions. Most proteins that bind PCNA contain a PCNA-interacting peptide (PIP) motif, a conserved motif containing only eight amino acids. Precisely how PCNA is able to discriminate between binding partners at the replication fork using only these small motifs remains unclear. Yeast CAF-1 contains a PIP motif on its largest subunit, Cac1. We solved the crystal structure of the PIP motif of CAF-1 bound to PCNA using a new strategy to produce stoichiometric quantities of one PIP motif bound to each monomer of PCNA. The PIP motif of CAF-1 binds to the hydrophobic pocket on the front face of PCNA in a similar manner to most known PIP-PCNA interactions. However, several amino acids immediately flanking either side of the PIP motif bind the IDCL or C-terminus of PCNA, as observed for only a couple other known PIP-PCNA interactions. Furthermore, mutational analysis suggests positively charged amino acids in these flanking regions are responsible for the low micromolar affinity of CAF-1 for PCNA, whereas the presence of a negative charge upstream of the PIP prevents a more robust interaction with PCNA. These results provide additional evidence that positive charges within PIP-flanking regions of PCNA-interacting proteins are crucial for specificity and affinity of their recruitment to PCNA at the replication fork.
Subject(s)
Chromatin Assembly Factor-1 , Models, Molecular , Proliferating Cell Nuclear Antigen , Protein Binding , Saccharomyces cerevisiae , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/genetics , Chromatin Assembly Factor-1/metabolism , Chromatin Assembly Factor-1/chemistry , Chromatin Assembly Factor-1/genetics , Crystallography, X-Ray , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Amino Acid Motifs , Binding Sites , Amino Acid Sequence , Protein ConformationABSTRACT
Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting superoxide (O2â-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). Human MnSOD has evolved to be highly product inhibited to limit the formation of H2O2, a freely diffusible oxidant and signaling molecule. The product-inhibited complex is thought to be composed of a peroxide (O22-) or hydroperoxide (HO2-) species bound to Mn ion and formed from an unknown PCET mechanism. PCET mechanisms of proteins are typically not known due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the mechanism, we combine neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states of the enzyme to reveal the positions of all the atoms, including hydrogen, and the electronic configuration of the metal ion. The data identifies the product-inhibited complex, and a PCET mechanism of inhibition is constructed.
Subject(s)
Superoxide Dismutase , Humans , Superoxide Dismutase/metabolism , Superoxide Dismutase/chemistry , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/chemistry , Manganese/metabolism , Manganese/chemistry , Electron Transport , Oxidation-Reduction , X-Ray Absorption Spectroscopy , Superoxides/metabolism , Superoxides/chemistry , Protons , Electrons , Models, Molecular , Oxygen/metabolism , Oxygen/chemistryABSTRACT
Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O 2 â¢- ) to molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pK a due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.
ABSTRACT
Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting O 2 â- to O 2 and H 2 O 2 with proton-coupled electron transfers (PCETs). Since changes in mitochondrial H 2 O 2 concentrations are capable of stimulating apoptotic signaling pathways, human MnSOD has evolutionarily gained the ability to be highly inhibited by its own product, H 2 O 2 . A separate set of PCETs is thought to regulate product inhibition, though mechanisms of PCETs are typically unknown due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the underlying mechanism, we combined neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states to reveal the all-atom structures and electronic configuration of the metal. The data identifies the product-inhibited complex for the first time and a PCET mechanism of inhibition is constructed.
ABSTRACT
Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting O2â- to O2 and H2O2 with proton-coupled electron transfers (PCETs). Since changes in mitochondrial H2O2 concentrations are capable of stimulating apoptotic signaling pathways, human MnSOD has evolutionarily gained the ability to be highly inhibited by its own product, H2O2. A separate set of PCETs is thought to regulate product inhibition, though mechanisms of PCETs are typically unknown due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the underlying mechanism, we combined neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states to reveal the all-atom structures and electronic configuration of the metal. The data identifies the product-inhibited complex for the first time and a PCET mechanism of inhibition is constructed.
ABSTRACT
The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain-Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)-γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV.
ABSTRACT
Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved. Here, we report the first crystal structure of full-length ClpP2 as an inactive homotetradecamer in a complex with a candidate antibiotic at 2.66 Å resolution. The structure details the functional domains of the ClpP2 protein subunit and includes the handle domain, which is integral to proteolytic activation. In addition, hydrogen-deuterium exchange mass spectroscopy probed the dynamics of ClpP2, and molecular modeling of ClpP1 predicted an assembly with ClpP2. By leveraging previous enzymatic experiments, we constructed a model of ClpP2 activation and its interaction with the protease subunits ClpP1 and ClpX. The structural information presented will be relevant for future rational drug design against these targets and will lead to a better understanding of ClpP complex formation and activation within this important human pathogen.
Subject(s)
Chlamydia trachomatis , Endopeptidase Clp , Models, Molecular , Humans , Anti-Bacterial Agents , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chlamydia trachomatis/enzymology , Endopeptidase Clp/chemistry , Endopeptidase Clp/metabolism , Crystallization , Protein DomainsABSTRACT
The COVID-19 pandemic caused by SARS-CoV-2 infection has led to socio-economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID-19. SARS-CoV-2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS-CoV-2 S spike expression and purification protocol from insect cells and studied four recombinant SARS-CoV-2 spike protein constructs based on the original SARS-CoV-2 sequence using a baculovirus expression system: a spike protein receptor-binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S-RBD-eGFP), spike ectodomain coupled to a fluorescent tag (S-Ecto-eGFP), spike ectodomain with six proline mutations and a foldon domain (S-Ecto-HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S-Ecto-HexaPro(-F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S-Ecto-eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Angiotensin-Converting Enzyme 2/genetics , Animals , Humans , Insecta/metabolism , Mammals , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Proline , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
The COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS-CoV-2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre-expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.
Subject(s)
SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Protein Domains , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.
Subject(s)
Homologous Recombination , Neoplasms/metabolism , Neoplasms/pathology , Rad52 DNA Repair and Recombination Protein/metabolism , Replication Protein A/metabolism , Apoptosis/drug effects , BRCA1 Protein/deficiency , BRCA1 Protein/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage , DNA Repair/drug effects , Doxorubicin/pharmacology , Fluorescence , High-Throughput Screening Assays , Homologous Recombination/drug effects , Humans , Mitoxantrone/pharmacology , Protein Binding/drug effects , Quinacrine/pharmacology , Small Molecule Libraries/pharmacologySubject(s)
Angiotensin-Converting Enzyme 2/metabolism , Bromelains/pharmacology , COVID-19 Drug Treatment , COVID-19 , SARS-CoV-2/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/metabolism , COVID-19/pathology , Chlorocebus aethiops , Humans , Vero CellsABSTRACT
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19. HIGHLIGHTS: Bromelain inhibits / cleaves the expression of ACE-2 and TMPRSS2Bromelain cleaves / degrades SARS-CoV-2 spike proteinBromelain inhibits S-Ectodomain binding and SARS-CoV-2 infection.
ABSTRACT
It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (BID, BIM, etc.) directly engage and allosterically activate effector proteins BAX/BAK. Here, through reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic BCL-2 proteins BCL-xL/MCL-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of BAX/BAK. BH3-only proteins' apoptotic activities correlate with affinities for BCL-xL/MCL-1 instead of abilities to directly activate BAX/BAK. Further, BID and BIM do not distinguish BAX from BAK or accelerate BAX/BAK activation following inactivation of BCL-xL/MCL-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and MCL-1 is fully restored by BID mutants capable of neutralizing BCL-xL, but not direct activation of BAX/BAK. Taken together, our findings provide a "Membrane-mediated Permissive" model, in which the BH3-only proteins only indirectly activate BAX/BAK by neutralizing the anti-apoptotic BCL-2 proteins, and thus allowing BAX/BAK to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation.
Subject(s)
Apoptosis/physiology , BH3 Interacting Domain Death Agonist Protein/physiology , Bcl-2-Like Protein 11/physiology , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , HCT116 Cells , HEK293 Cells , Humans , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/metabolismABSTRACT
Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients' response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.