Michael J. McGuire

Recent advances in electronic structure theory: Method of moments of coupled-cluster equations and renormalized coupled-cluster approaches

The recently developed new approach to the many-electron correlation problem in atoms and molecules, termed the method of moments of coupled-cluster (CC) equations (MMCC), is reviewed. The ground-state MMCC formalism and its extension to excited electronic states via the equation-of-motion coupled-cluster (EOMCC) approach are discussed. The main principle of all MMCC methods is that of the non-iterative energy corrections which, when added to the ground- and excited-state energies obtained in the standard CC calculations, such as CCSD or EOMCCSD, recover the exact, full configuration interaction (CI) energies. Three types of the MMCC approximations are reviewed in detail: (i) the CI-corrected MMCC methods, which can be applied to ground and excited states; (ii) the renormalized and completely renormalized CC methods for ground states; and (iii) the quasi-variational MMCC approaches for the ground-state problem, including the quadratic MMCC models. It is demonstrated that the MMCC formalism provides a new theoretical framework for designing black-box CC approaches that lead to an excellent description of entire potential energy surfaces of ground- and excited-state molecular systems with an ease of use of the standard single-reference methods. The completely renormalized (CR) CCSD(T) and CCSD(TQ) methods and their quadratic and excited-state MMCC analogues remove the failing of the standard CCSD, CCSD(T), EOMCCSD and similar methods at larger internuclear separations and for states that normally require a genuine multireference description. All theoretical ideas are illustrated by numerical examples involving bond breaking, excited vibrational states, reactive potential energy surfaces and difficult cases of excited electronic states. The description of the existing and well-established variants of the MMCC theory, such as CR-CCSD(T), is augmented by the discussion of future prospects and potentially useful recent developments, including the extension of the black-box CR-CCSD(T) method to excited states.

Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands

One limitation in the development of biosensors for the early detection of disease is the availability of high specificity and affinity ligands for biomarkers that are indicative of a pathogenic process. Within the past 10 years, biopanning of phage displayed peptide libraries on intact cells has proven to be a successful route to the identification of cell-specific ligands. The peptides selected from these combinatorial libraries are often able to distinguish between diseased cells and their normal counterparts as well as cells in different activation states. These ligands are small and chemical methodologies are available for regiospecific derivatization. As such, they can be incorporated into a variety of different diagnostic and therapeutic platforms. Here we describe the methods utilized in the selection of peptides from phage displayed libraries by biopanning. In addition, we provide methods for the synthesis of the selected peptides as both monomers and tetramers. Downstream uses for the peptides are illustrated.

A Peptide Selected by Biopanning Identifies the Integrin αvβ6 as a Prognostic Biomarker for Nonsmall Cell Lung Cancer

The development of new modes of diagnosis and targeted therapy for lung cancer is dependent on the identification of unique cell surface features on cancer cells and isolation of reagents that bind with high affinity and specificity to these biomarkers. We recently isolated a 20-mer peptide which binds to the lung adenocarcinoma cell line, H2009, from a phage-displayed peptide library. We show here that the cellular receptor for this peptide, TP H2009.1, is the uniquely expressed integrin, αvβ6, and the peptide binding to lung cancer cell lines correlates to integrin expression. The peptide is able to mediate cell-specific uptake of a fluorescent nanoparticle via this receptor. Expression of αvβ6 was assessed on 311 human lung cancer samples. The expression of this integrin is widespread in early-stage nonsmall cell lung carcinoma (NSCLC). Log-rank test and Cox regression analyses show that expression of this integrin is significantly associated with poor patient outcome. Preferential expression is observed in the tumors compared with the surrounding normal lung tissue. Our data indicate that αvβ6 is a prognostic biomarker for NSCLC and may serve as a receptor for targeted therapies. Thus, cell-specific peptides isolated from phage biopanning can be used for the discovery of cell surface biomarkers, emphasizing the utility of peptide libraries to probe the surface of a cell. [Cancer Res 2007;67(12):5889–95]

The high molecular weight multicatalytic proteinase, macropain, exists in a latent form in human erythrocytes

The high molecular weight multicatalytic proteinase, macropain, has been purified from human erythrocytes in two forms that differ in caseinolytic activity up to 100-fold. Each form has a native molecular weight of 600,000 and is composed of a number of subunits ranging in molecular weights from 35,000 to 21,000. Although the two proteinase forms share a number of electrophoretically indistinguishable subunits, there are also subunits unique to the respective forms. The less active proteinase represents a latent enzyme because it was fully activated by two procedures including dialysis against water and pretreatment with low concentrations of sodium dodecyl sulfate. These procedures caused differential changes in the caseinolytic and two peptidase activities of the proteinase. An Mr 35,000 subunit, characteristic of latent macropain, is immunologically related to at least one of the other components of active macropain and disappeared after proteinase activation by dialysis. Nevertheless, loss of this subunit was not the cause of the increased activity. These results suggest that the proteolytic activity of cells may be regulated by the activation of the latent form of macropain.

Purification and characterization of dipeptidyl peptidase I from human spleen

The lysosomal hydrolase, dipeptidyl peptidase I (DPPI), was purified from human spleen and its enzymatic activity characterized. The enzyme was purified to apparent homogeneity by a combination of differential pH solubility, heat-treatment, affinity chromatography on concanavalin A-agarose and p-hydroxymercuribenzoate-agarose, and gel filtration chromatography on Sephacryl S-300. This procedure resulted in a 1100-fold purification of DPPI protein with a yield of approximately 2% of the total DPPI activity. The enzyme was characterized as a glycoprotein with a pI of 5.4, a molecular mass of 200,000 Da as determined by gel filtration under nondenaturing conditions, and a subunit size of 24,000 Da. Amino acid sequence analysis of peptides isolated from cyanogen bromide and trypsin digests of the 24,000-Da subunit revealed extensive sequence similarity between human and rat DPPI. Purified DPPI exhibited both hydrolytic and transpeptidase (polymerase) activity. DPPI exhibited activity against a variety of dipeptide substrates including peptides with either non-polar or polar residues in the P1 position. In contrast to the reported substrate specificity of bovine and murine DPPI, the human enzyme exhibited a modest preference for peptides with nonpolar residues in the P1 position. DPPI content was found to be highest among cytotoxic lymphocytes and myeloid cells. The high level of DPPI expression in these cell populations correlates with their sensitivity to the toxic effects of leucyl-leucine methyl ester, a substrate for DPPI.

Relationships among the subunits of the high molecular weight proteinase, macropain (proteasome)

An analysis of the subunits of the high molecular weight proteinase, macropain (multicatalytic proteinase or proteasome) from human erythrocytes has been conducted using N-terminal amino acid sequencing, gel electrophoresis and reverse-phase peptide mapping. This analysis provided evidence for the existence of 13 subunits of different primary structure. Five subunits were susceptible to the Edman degradation and yielded unique N-terminal sequences. Similarities among these sequences, however, indicated that the subunits are homologous. Two-dimensional gel electrophoresis discriminated 10 major components, which included two of the subunits for which N-terminal sequences had been determined and eight N-terminally blocked subunits. Tryptic peptide mapping indicated that all 10 of these components have a different amino acid sequence. Tryptic peptides from some of the subunits were subjected to amino acid sequence analysis, and the data indicated that all the subunits tested in this way are related by common ancestry. The data suggest that at least nine of the total of 13 subunits are encoded by members of the same gene family; the remaining four subunits have not yet been investigated in sufficient detail to establish their relationships. No evidence for a close relationship with any previously investigated proteinase family has been found. Finally, through a comparison of the latent and active forms of macropain, the study established a close similarity in the subunit composition of these catalytically very different species, although proteolytic degradation of selected subunits appears in the active form of the enzyme.

Peptide-Targeted Polyglutamic Acid Doxorubicin Conjugates for the Treatment of αvβ6-Positive Cancers

Most chemotherapeutics exert their effects on tumor cells as well as their healthy counterparts, resulting in dose limiting side effects. Cell-specific delivery of therapeutics can increase the therapeutic window for treatment by maintaining the therapeutic efficacy while decreasing the untoward side effects. We have previously identified a peptide, named H2009.1, which binds to the integrin alpha(v)beta(6). Here, we report the synthesis of a peptide targeted polyglutamic acid polymer in which the high affinity alpha(v)beta(6)-specific tetrameric H2009.1 peptide is incorporated via a thioether at the N-terminus of a 15 amino acid polymer of glutamic acid. Doxorubicin is incorporated into the polymer via an acid-labile hydrazone bond. Payloads of four doxorubicin molecules per targeting agent are achieved. The drug is released at pH 4.0 and 5.6 but the conjugate is stable at pH 7.0. The conjugate is selectively internalized into alpha(v)beta(6) positive cells as witnessed by flow cytometric analysis and fluorescent microscopy. Cellular uptake is mediated by the H2009.1 peptide, as no internalization of the doxorubicin-PG polymer is observed when it is conjugated to a scrambled sequence control peptide. Importantly, the conjugate is more cytotoxic toward a targeted cell than a cell line that does not express the integrin.

An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.

Cloning and characterization of the cDNA encoding mouse dipeptidyl peptidase I (Cathepsin C)

The mouse dipeptidyl peptidase I cDNA has been cloned and sequenced and patterns of DPPI mRNA expression in various tissues characterized. Sequences encoding DPPI are highly conserved among mouse, rat and human and include an unusually long propeptide and a distinctive tyrosine adjacent to the active site cysteine.

Genetic Immunization ☆: What's in a Name?

The concept and demonstration of genetic immunization (GI) was first introduced in 1992. At the time it appeared to be a revolutionary new approach in vaccinology. Since then, genetic immunization has been applied with much success in a wide variety of model and natural systems. It has also been used in several human clinical trials. Currently there is a general impression that genetic immunization has limitations inhibiting its broad use. The technique is thought to be poor at antibody production and more importantly not to work well in primates and humans (simian barrier). However, recent reports addressing these issues (poor antibody production and the simian barrier) showed improvements of GI to produce protective immune responses in humans. We propose that the apparent limitations of gene vaccines may arise from not using the technologies potential to manipulate the immune system. This dearth of imaginative use is manifested in the tendency by some to term the technique DNA immunization. The apparent limitations of DNA vaccines may not be limitations for gene vaccines.