Shelly Pizarro - Academia.edu (original) (raw)

Papers by Shelly Pizarro

The Journal of biological chemistry, Jan 25, 1993

Shortly after activation by either thrombin or the tethered ligand domain peptide SFLLRN, thrombi... more Shortly after activation by either thrombin or the tethered ligand domain peptide SFLLRN, thrombin receptors undergo homologous desensitization, temporarily losing their ability to respond to both agonists. We have examined the role of receptor internalization and recycling in this process using receptor-directed antibodies as probes. The results show within 1 min of activation > 85% of the approximately 200,000 thrombin receptors on megakaryoblastic human erythroleukemia (HEL) and CHRF-288 cells are sequestered into endosomes via coated pits, after which the majority are transferred to lysosomes. This process does not require proteolysis of the receptor and occurs with sufficient speed to play a major role in the regulation of thrombin receptor function. Although most of the internalized receptors are ultimately degraded, approximately 25% return to the cell surface. These recycled receptors are in a state in which they can respond to SFLLRN but not thrombin; nor do they self-ac...

Seminars in hematology, 1994

Recent studies have helped to define the mechanisms by which thrombin activates platelets and oth... more Recent studies have helped to define the mechanisms by which thrombin activates platelets and other cells. Those studies show that the human thrombin receptor has a structure similar to other G protein-coupled receptors, but is activated by a novel mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Shortly after activation, thrombin receptors become temporarily resistant to re-activation. Present evidence suggests that this loss of function is due to a combination of receptor desensitization, phosphorylation and internalization, and that recovery may involve dephosphorylation, as well as receptor recycling and the expression of newly-synthesized receptors. Together these processes provide a potent mechanism for limiting the duration of thrombin-initiated events in platelets and other thrombin-responsive vascular cells.

The Journal of biological chemistry, Jan 28, 1994

According to current models, human thrombin receptors are activated when thrombin cleaves the rec... more According to current models, human thrombin receptors are activated when thrombin cleaves the receptor's N terminus, exposing the tethered ligand domain, SFLLRN. In the megakaryoblastic CHRF-288 cell line, thrombin receptor activation is followed by the rapid internalization of > 90% of the receptors. In the present studies, antibodies directed at the site of cleavage by thrombin were used to examine changes in receptor structure during activation, internalization, and recovery. As would be expected, the initial rate of receptor cleavage was directly related to the thrombin concentration. However, even after prolonged incubation, receptor cleavage was incomplete until the thrombin concentration exceeded the receptor concentration. Only cleaved receptors were internalized in response to thrombin and only catalytically active thrombin and active variants of SFLLRN-containing peptides caused receptor internalization. Over a 3-h period following receptor activation by thrombin, t...

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, Jan 15, 2014

This proof-of-concept study examines the applicability of using multimodal chromatography to sele... more This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development. A range of dynamic binding capacities was observed for 10 feedstocks (24-53g/L) and two feedstocks had poor binding properties (<10g/L) despite load conditioning towards a more favorable pH. Analysis of the DBC versus molecular properties showed that the mAb-ligand binding interaction was predominantly charge based. Four separa...

Journal of The American Chemical Society - J AM CHEM SOC, 2000

Protein Expression and Purification, 2010

Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and ... more Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

Physical Chemistry Chemical Physics, 2004

X-Ray spectroscopy is used to examine the effect of the manganese oxidation state for a series of... more X-Ray spectroscopy is used to examine the effect of the manganese oxidation state for a series of Mn model compounds. Sensitive to Mn oxidation and structural symmetry, X-ray absorption and emission spectroscopy (XAS and XES) provide complementary insights. However, few benchmark examples of complexes with similar structures but in different oxidation states are available to evaluate data from unknown structures like the oxygen evolving complex (OEC) of Photosystem II (PSII). This study examines two types of compounds prepared in a variety of Mn oxidation states and which possess chemical structures with Mn-Mn interactions (~2.7 Å and ~3.3 Å) that have been observed in the OEC. Model complexes with core compositions Mn3O and Mn4O2 contain combinations of Mn in either a reduced (II) or oxidized (III) state. Within each set of compounds, complexes with higher Mn oxidation states have absorption K-edge energy values that are higher (1.6-2.2 eV) than those of their more reduced counterparts. This trend is accordingly reversed in the Kβ emission spectroscopy where the first moment energy values are lower (0.09-0.12 eV) for compounds with higher Mn oxidation states. We will discuss in detail, how these trends can be quantitatively used to characterize the effects of the Mn oxidation state as well as the surrounding ligand environment on the observed X-ray spectra. The results are discussed with respect to previously obtained data on different S-states of the OEC.

Journal of the American Chemical Society, 2002

A key component required for an understanding of the mechanism of the evolution of molecular oxyg... more A key component required for an understanding of the mechanism of the evolution of molecular oxygen by the photosynthetic oxygen-evolving complex (OEC) in photosystem II (PS II) is the knowledge of the structures of the Mn cluster in the OEC in each of its intermediate redox states, or S-states. In this paper, we report the first detailed structural characterization using Mn extended X-ray absorption fine structure (EXAFS) spectroscopy of the Mn cluster of the OEC in the S(0) state, which exists immediately after the release of molecular oxygen. On the basis of the EXAFS spectroscopic results, the most likely interpretation is that one of the di-mu-oxo-bridged Mn-Mn moieties in the OEC has increased in distance from 2.7 A in the dark-stable S(1) state to 2.85 A in the S(0) state. Furthermore, curve fitting of the distance heterogeneity present in the EXAFS data from the S(0) state leads to the intriguing possibility that three di-mu-oxo-bridged Mn-Mn moieties may exist in the OEC instead of the two di-mu-oxo-bridged Mn-Mn moieties that are widely used in proposed structural models for the OEC. This possibility is developed using novel structural models for the Mn cluster in the OEC which are consistent with the structural information available from EXAFS and the recent X-ray crystallographic structure of PS II at 3.8 A resolution.

Journal of the American Chemical Society, 2001

A key question for the understanding of photosynthetic water oxidation is whether the four oxidiz... more A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S(3) --> [S(4)] --> S(0) transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kbeta X-ray emission spectroscopy (Kbeta XES) to this problem for the first time. The Kbeta XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S(2) --> S(3) transition, in contrast to the S(0) --> S(1) and S(1) --> S(2) transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S(3)-state, manganese mu-oxo bridge radical formation is proposed for the S(2) --> S(3) transition, and three possible mechanisms for the O-O bond formation are presented.

Journal of Synchrotron Radiation, 2001

The combination of large-acceptance high-resolution X-ray optics with bright synchrotron sources ... more The combination of large-acceptance high-resolution X-ray optics with bright synchrotron sources permits quantitative analysis of rare events such as X-ray¯uorescence from very dilute systems, weak uorescence transitions or X-ray Raman scattering. Transition-metal K¯uorescence contains information about spin and oxidation state; examples of the characterization of the Mn oxidation states in the oxygen-evolving complex of photosystem II and Mn-consuming spores from the marine bacillus SG-1 are presented. Weaker features of the K spectrum resulting from valence-level and`interatomic' ligand to metal transitions contain detailed information on the ligandatom type, distance and orientation. Applications of this spectral region to characterize the local structure of model compounds are presented. X-ray Raman scattering (XRS) is an extremely rare event, but also represents a unique technique to obtain bulk-sensitive lowenergy (<600 eV) X-ray absorption ®ne structure (XAFS) spectra using hard ($10 keV) X-rays. A photon is inelastically scattered, losing part of its energy to promote an electron into an unoccupied level. In many cases, the cross section is proportional to that of the corresponding absorption process yielding the same X-ray absorption near-edge structure (XANES) and extended X-ray absorption ®ne structure (EXAFS) features. XRS ®nds application for systems that defy XAFS analysis at low energies, e.g. liquids or highly concentrated complex systems, reactive compounds and samples under extreme conditions (pressure, temperature). Recent results are discussed.

Journal of Chromatography A, 2010

Mixed-mode chromatography resins are gaining popularity as effective purification tools for chall... more Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with &amp;gt;95% yields while reducing host cell protein content to &amp;lt;1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.

Journal of Biological Inorganic Chemistry, 2004

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OE... more Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn-Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures Mn(IV)(3)O(4)X (X=Cl, F, or OH) that contain a di-micro-oxo and two mono-micro-oxo bridges or Mn(IV)(2)O(2)X (X=Cl, F, OH, OAc) that contain a di-micro-oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn-X interactions (X=O, N, Cl, F), and the best fits were found when a Mn-Cl interaction at a 2.2-2.3 A bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.

ELECTROPHORESIS, 2007

A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip g... more A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip gel electrophoresis (CGE) coupled with LIF detection was used to analyze lysates from different bacterial cell lines to obtain signature profiles of the soluble protein composition. The study includes Escherichia coli, Bacillus subtilis, and Bacillus anthracis (Delta Sterne strain) vegetative cells as well as endospores formed from the latter two species as model organisms to demonstrate the method. A unified protein preparation protocol was developed for both cell types to streamline the benchtop process and aid future automation. Cells and spores were lysed and proteins solubilized using a combination of thermal and chemical lysis methods. Reducing agents, necessary to solubilize spore proteins, were eliminated using a small-scale rapid size-exclusion chromatography step to eliminate interference with down-stream protein labeling. This approach was found to be compatible with nonspore cells (i.e., vegetative cells) as well, not adversely impacting the protein signatures. Data are presented demonstrating distinct CGE protein signatures for our model organisms, suggesting the potential for discrimination of organisms on the basis of empirical protein patterns. The goal of this work is to develop a fast and field-portable method for characterizing bacteria via their proteomes.

Biotechnology Progress, 2011

High-pressure has been established as an effective technique for refolding proteins at high conce... more High-pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1-3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large-scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29-42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high-pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high-pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale-up for this molecule.

Biotechnology and Bioengineering, 2009

Process analytical technology (PAT) is an initiative from the US FDA combining analytical and sta... more Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small-scale bioreactor (3 L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse-phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (k(L)a) is used for scale-up of the aeration parameters to manufacturing scale tanks (2,000 L, 15,000 L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to &amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;actively manage process variability using a knowledge-based approach.&amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;

Biochemistry, 2002

The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrat... more The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion. The new Ca EXAFS clearly shows backscattering from Mn at 3.4 A, a distance that agrees with that surmised from previously recorded Mn EXAFS. This result is also consistent with earlier related experiments at the Sr K-edge, using samples that contained functional Sr, that show Mn is approximately 3.5 A distant from Sr. The totality of the evidence clearly advances the notion that the catalytic center of oxygen evolution is a Mn-Ca heteronuclear cluster.

Annals of the New York Academy of Sciences, 1994

BRASS, LF, AHUJA, M., BELMONTE, E., PIZARRO, S., TARVER, A. and HOXIE, JA (1994), The Human Plate... more BRASS, LF, AHUJA, M., BELMONTE, E., PIZARRO, S., TARVER, A. and HOXIE, JA (1994), The Human Platelet Thrombin Receptor. Annals of the New York Academy of Sciences, 714: 112. doi: 10.1111/j. 1749-6632.1994. tb12025. x

Photochemistry and Photobiology, 2001

C-Phycocyanin (PC) trimers associated with linker polypeptides were isolated from the phycobiliso... more C-Phycocyanin (PC) trimers associated with linker polypeptides were isolated from the phycobilisome (PBS) rods of Synechococcus sp. PCC 7002. L X Y refers to a linker polypeptide (L) having an apparent mass of Y kDa, located at position X in the phycobilisome where X can be R (rod), C (core) or RC (rod-core junction). Measurements of the absorption, fluorescence and excitation anisotropy of PC trimer, PC·L R 32.3 and PC·L RC 28.5 complexes document the spectroscopic modulation of each linker polypeptide on the PC chromophores. The difference spectra between the PC trimer and the PC-linker complexes show that although the effect induced by the linker polypeptides is qualitatively similar in behavior, the extent of the modulation is greater in PC·L RC 28.5 . Measurements taken at 77 K show that a red-wavelength component of the PC trimer absorption-fluorescence spectra is the target of the linker's influence and that this component is altered to a greater extent by L RC 28.5 . In addition the 77 K absorbance of the PC trimer resolves band features that are consistent with an excitonic coupling interaction between neighboring ␣84 and ␤84 chromophores. These band features are also evident in the absorbance of PC·L R 32.3 but are absent in PC·L RC 28.5 indicating that L RC 28.5 may be perturbing the coupling interaction established in the PC trimer ␣84-␤84 chromophore pairs. Structurally, the linker polypeptide should disrupt the C 3 symmetry in the central cavity of the associated phycobiliprotein and this asymmetric interaction should serve to guide the transfer of excitation energy along PBS rods toward the core elements.