Papers by Alexander Shekhtman
Proceedings of the National Academy of Sciences, 2004
Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, t... more Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The 1 J NC′ coupling constant for the (-1) scissile peptide bond at the N-extein–intein junction was found to be ≈12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (-1) scissile bond in its unusual conformation. Overall, these studies s...
A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins
Biochemical and Biophysical Research Communications, 2002
The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, w... more The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, which is present in, but not limited to, the proteins involved in endocytotic and proteasomal degradation. UIMs facilitate endocytotic vesicular sorting of the monoubiquitinated proteins and may be important for the targeting of the polyubiquitinated proteins to the proteasome. Using heteronuclear NMR backbone and side-chain chemical shift mapping of the ubiquitin interaction surface, the UIM from the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, specifically binds to the ubiquitin hydrophobic surface using UIM's well-conserved central helical LALAL motif. Molecular modeling of the ubiquitin:UIM_Hrs complex suggests that binding occurs through a specific interaction of Leu263 and Leu267 of the UIM_Hrs with two ubiquitin hydrophobic patches located in close proximity to the ubiquitin major polyubiquitination site, Lys48. Intramolecular binding of ubiquitin to a UIM in monoubiquitinated proteins would render Lys48 unavailable for further ubiquitination, thus, explaining the absolute requirement of UIMs for monoubiquitination. Two leucines, Leu265 and Leu269, located on the opposite face of UIM_Hrs can also interact, albeit less favorably than Leu263 and Leu267, with the ubiquitin hydrophobic patches, suggesting a possible mode for polyubiquitin:UIM binding and apparent preference of UIMs for polyubiquitins.
Physics Letters A, 1996
A strong electromagnetic field exerts a force on neutral atoms or molecules and this effect has b... more A strong electromagnetic field exerts a force on neutral atoms or molecules and this effect has been utilized in atomic beam optics to create the analogs to optical elements such as lenses and mirrors. We extend this concept to the specific interaction of a chiral molecule with a circularly polarized laser wave. Because of the optical activity of chiral molecules this interaction is selective with respect to handedness and this selectivity forms the basis for a "chiral mirror" scheme, here introduced, to produce chirally pure matter.
Communications Biology
High-resolution structural studies of proteins and protein complexes in a native eukaryotic envir... more High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin.
International Journal of Molecular Sciences
Increasing evidence links the RAGE (receptor for advanced glycation end products)/DIAPH1 (Diaphan... more Increasing evidence links the RAGE (receptor for advanced glycation end products)/DIAPH1 (Diaphanous 1) signaling axis to the pathogenesis of diabetic complications. RAGE is a multi-ligand receptor and through these ligand–receptor interactions, extensive maladaptive effects are exerted on cell types and tissues targeted for dysfunction in hyperglycemia observed in both type 1 and type 2 diabetes. Recent evidence indicates that RAGE ligands, acting as damage-associated molecular patterns molecules, or DAMPs, through RAGE may impact interferon signaling pathways, specifically through upregulation of IRF7 (interferon regulatory factor 7), thereby heralding and evoking pro-inflammatory effects on vulnerable tissues. Although successful targeting of RAGE in the clinical milieu has, to date, not been met with success, recent approaches to target RAGE intracellular signaling may hold promise to fill this critical gap. This review focuses on recent examples of highlights and updates to the...
Current Cardiology Reports
Purpose of Review The cardiovascular complications of type 1 and 2 diabetes are major causes of m... more Purpose of Review The cardiovascular complications of type 1 and 2 diabetes are major causes of morbidity and mortality. Extensive efforts have been made to maximize glycemic control; this strategy reduces certain manifestations of cardiovascular complications. There are drawbacks, however, as intensive glycemic control does not impart perennial protective benefits, and these efforts are not without potential adverse sequelae, such as hypoglycemic events. Recent Findings Here, the authors have focused on updates into key areas under study for mechanisms driving these cardiovascular disorders in diabetes, including roles for epigenetics and gene expression, interferon networks, and mitochondrial dysfunction. Updates on the cardioprotective roles of the new classes of hyperglycemia-targeting therapies, the sodium glucose transport protein 2 inhibitors and the agonists of the glucagon-like peptide 1 receptor system, are reviewed. Summary In summary, insights from ongoing research and t...
PLOS ONE
Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and mo... more Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.
Immunometabolism, 2021
Fundamental modulation of energy metabolism in immune cells is increasingly being recognized for ... more Fundamental modulation of energy metabolism in immune cells is increasingly being recognized for the ability to impart important changes in cellular properties. In homeostasis, cells of the innate immune system, such as monocytes, macrophages and dendritic cells (DCs), are enabled to respond rapidly to various forms of acute cellular and environmental stress, such as pathogens. In chronic stress milieus, these cells may undergo a reprogramming , thereby triggering processes that may instigate tissue damage and failure of resolution. In settings of metabolic dysfunction, moieties such as excess sugars (glucose, fructose and sucrose) accumulate in the tissues and may form advanced glycation end products (AGEs), which are signaling ligands for the receptor for advanced glycation end products (RAGE). In addition, cellular accumulation of cholesterol species such as that occurring upon macrophage engulfment of dead/dying cells, presents these cells with a major challenge to metabolize/efflux excess cholesterol. RAGE contributes to reduced expression and activities of molecules mediating cholesterol efflux. This Review chronicles examples of the roles that sugars and cholesterol, via RAGE, play in immune cells in instigation of maladaptive cellular signaling and the mediation of chronic cellular stress. At this time, emerging roles for the ligand-RAGE axis in metabolism-mediated modulation of inflammatory signaling in immune cells are being unearthed and add to the growing body of factors underlying pathological immunometabolism.
Communications Biology, 2020
Protein–protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a p... more Protein–protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real time in-cell NMR spectroscopy, RT-NMR, utilizes a bioreactor to maintain cells in an active metabolic state. Improvement in bioreactor technology maintains ATP levels at >95% for up to 24 hours, enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected. Singular value decomposition, SVD, of the NMR spectra collected over the course of Mpa overexpression easily identified the PPIs despite the large variation in background signals due to the highly active metabolome.
Methods in Enzymology, 2019
In-cell NMR spectroscopy is a powerful tool to study protein structures and interactions under ne... more In-cell NMR spectroscopy is a powerful tool to study protein structures and interactions under near physiological conditions in both prokaryotic and eukaryotic living cells. The low sensitivity and resolution of in-cell NMR spectra and limited lifetime of cells over the course of an in-cell experiment have presented major hurdles to wide acceptance of the technique, limiting it to a few select systems. These issues are addressed by introducing the use of the CRINEPT pulse sequence to increase the sensitivity and resolution of in-cell NMR spectra and the use of a bioreactor to maintain cell viability for up to 24 h. Application of advanced pulse sequences and bioreactor during in-cell NMR experiments will facilitate the exploration of a wide range of biological processes.
ACS Chemical Biology, 2018
In-cell NMR spectroscopy was used to screen for drugs that disrupt the interaction between prokar... more In-cell NMR spectroscopy was used to screen for drugs that disrupt the interaction between prokaryotic ubiquitin like protein, Pup, and mycobacterial proteasome ATPase, Mpa. This interaction is critical for Mycobacterium tuberculosis resistance against nitric oxide (NO) stress; interruption of this process was proposed as a mechanism to control latent infection. Three compounds isolated from the NCI Diversity set III library rescued the physiological proteasome substrate from degradation suggesting that the proteasome degradation pathway was selectively targeted. Two of the compounds bind to Mpa with sub-micromolar to nanomolar affinity, and all three exhibit potency toward mycobacteria comparable to antibiotics currently available on the market, inhibiting growth in the low micromolar range.
Biochemistry, 2018
It is not well understood how ribosome antibiotics affect a wide range of biochemical pathways; c... more It is not well understood how ribosome antibiotics affect a wide range of biochemical pathways; changes in RNA-mediated protein quinary interactions and consequent activity inside the crowded cytosol may provide one possible mechanism. We developed real-time (RT) in-cell NMR spectroscopy to monitor temporal changes in protein quinary structure, for 24 hours and longer, in response to external and internal stimuli. RT in-cell NMR consists of a bioreactor containing gel encapsulated cells inside a 5 mm NMR tube, a gravity siphon for continuous exchange of medium, and a horizontal drip irrigation system to supply nutrients to the cells during the experiment. We showed that adding antibiotics that bind to the small ribosomal subunit result in more extensive quinary interactions between thioredoxin and mRNA. The results substantiate the idea that RNAmediated modulation of quinary protein interactions may provide the physical basis for ribosome inhibition and other regulatory pathways.
Bioorganic & Medicinal Chemistry, 2017
We report the high-yield heterologous expression of bioactive θ-defensin RTD-1 inside Escherichia... more We report the high-yield heterologous expression of bioactive θ-defensin RTD-1 inside Escherichia coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. RTD-1 is a small backbone-cyclized polypeptide with three disulfide bridges and a natural inhibitor of anthrax lethal factor protease. Recombinant RTD-1 was natively folded and able to inhibit anthrax lethal factor protease. In-cell expression of RTD-1 was very efficient and yielded ≈ 0.7 mg of folded RTD-1 per gram of wet E. coli cells. This approach was used to generate of a genetically-encoded RTD-1-based peptide library in live E. coli cells. These results clearly demonstrate the possibility of using genetically-encoded RTD-1-based peptide libraries in live E. coli cells, which is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide RTD-1 as a molecular scaffold.
Biochemistry, Jan 15, 2017
Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of... more Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of the cell volume. Under these conditions, even weak quinary interactions between ribosomes and cytosolic proteins can affect protein activity. By using in-cell and in vitro NMR spectroscopy, and biophysical techniques, we show that the enzymes, adenylate kinase and dihydrofolate reductase, and the respective coenzymes, ATP and NADPH, bind to ribosomes with micromolar affinity, and that this interaction suppresses the enzymatic activities of both enzymes. Conversely, thymidylate synthase, which works together with dihydrofolate reductase in the thymidylate synthetic pathway, is activated by ribosomes. We also show that ribosomes impede diffusion of green fluorescent protein in vitro and contribute to the decrease in diffusion in vivo. These results strongly suggest that ribosome-mediated quinary interactions contribute to the differences between in vitro and in vivo protein activities and...
Molecular microbiology, Jan 2, 2017
Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling networ... more Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macropha...
Inorganic chemistry, Jan 3, 2017
Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein comple... more Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagneti...
Journal of medicinal chemistry, Jan 9, 2017
θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC50 = 390... more θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC50 = 390 ± 20 nM, Ki = 365 ± 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNFα converting enzyme (TACE) (Ki = 4.45 ± 0.48 μM). Using full sequence amino acid scanning in combination with a highly efficient "one-pot" cyclization-folding approach, we obtained an RTD-1-based peptide that was around 10 times more active than wild-type RTD-1 in inhibiting LF protease (IC50 = 43 ± 3 nM, Ki = 18 ± 1 nM). The most active peptide was completely symmetrical, rich in Arg and Trp residues, and able to adopt a native RTD-1-like structure. These results show the power of optimized chemical peptide synthesis approaches for the efficient production of libraries of disulfide-rich backbone-cyclized peptides to quickly perform structure-activity relationship studies for optimizing protease inhibitors.
Journal of inorganic biochemistry, May 24, 2017
Paramagnetic resonance enhancement (PRE) is an NMR technique that allows studying three-dimension... more Paramagnetic resonance enhancement (PRE) is an NMR technique that allows studying three-dimensional structures of RNA-protein complexes in solution. RNA strands are typically spin labeled using nitroxide reagents, which provide minimal perturbation to the native structure. The current work describes an alternative approach, which is based on a Co(2+)-based probe that can be covalently attached to RNA in the vicinity of the protein's binding site using 'click' chemistry. Similar to nitroxide spin labels, the transition metal based probe is capable of attenuating NMR signal intensities from protein residues localized <40Å away. The extent of attenuation is related to the probe's distance, thus allowing for construction of the protein's contact surface map. This new paradigm has been applied to study binding of HIV-1 nucleocapsid protein 7, NCp7, to a model RNA pentanucleotide.
Biochemistry, 2016
RNA constitutes up to 20% of a cell's dry weight, corresponding to ~20 mg/mL. This high concentra... more RNA constitutes up to 20% of a cell's dry weight, corresponding to ~20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states.
Structure (London, England : 1993), Sep 9, 2016
The weak oligomerization exhibited by many transmembrane receptors has a profound effect on signa... more The weak oligomerization exhibited by many transmembrane receptors has a profound effect on signal transduction. The phenomenon is difficult to characterize structurally due to the large sizes of and transient interactions between monomers. The receptor for advanced glycation end products (RAGE), a signaling molecule central to the induction and perpetuation of inflammatory responses, is a weak constitutive oligomer. The RAGE domain interaction surfaces that mediate homo-dimerization were identified by combining segmental isotopic labeling of extracellular soluble RAGE (sRAGE) and nuclear magnetic resonance spectroscopy with chemical cross-linking and mass spectrometry. Molecular modeling suggests that two sRAGE monomers orient head to head forming an asymmetric dimer with the C termini directed toward the cell membrane. Ligand-induced association of RAGE homo-dimers on the cell surface increases the molecular dimension of the receptor, recruiting Diaphanous 1 (DIAPH1) and activatin...
Uploads
Papers by Alexander Shekhtman