Acquisition of inhibitor-sensitive protein kinases through protein design.

Protein phosphorylation is the major post-translational modification used by eukaryotic cells to control cellular signaling. Protein kinases have emerged as attractive drug targets because heightened protein kinase activity has been associated with several proliferative diseases, most notably cancer and restenosis. Until now, it has been very difficult to confirm the utility of protein kinases as inhibitor targets because very few small molecules that selectively inhibit one particular kinase are known. Discovery of highly specific kinase inhibitors has been slow because the protein family contains approximately 2000 members, all of which share a conserved active site fold. Recent work in several laboratories has sought to circumvent the problem of kinase structural degeneracy by engineering drug sensitivity into Src family tyrosine kinases and mitogen-activated protein kinases through site-directed mutagenesis. By introducing a unique non-naturally occurring amino acid into a conserved region of the enzyme's binding site, a target protein kinase can be rapidly sensitized to a small molecule. Introduction of the engineered kinase into a cell line or animal model should greatly expedite the investigation of protein kinase inhibition as a viable drug treatment. The purpose of this review is to summarize these recent advances in protein kinase drug sensitization.     

POE-PEG-POE triblock copolymeric microspheres containing protein. II. Polymer erosion and protein release mechanism.

The first paper of this series presented the fabrication and characterization of POE-PEG-POE triblock copolymeric microspheres containing protein. In this paper, we focus on the polymer erosion and the mechanism of protein release. Fourteen-week in vitro behaviors of POE-PEG-POE microspheres loaded with bovine serum albumin (BSA) have been monitored. SEM micrographs reveal that after 14-week incubation in PBS buffer, pH 7.4, 37 degrees C, the polymeric particles remain spherical despite mass loss of almost 90%. On the other hand, molecular weight undergoes a high initial loss of 38% and 44% during the first 2-week incubation for POE-PEG(5%)-POE and POE-PEG(10%)-POE, respectively. Then, it keeps relatively unchanged over 12 weeks. However, POE-PEG(20%)-POE copolymer provides a better compatibility between the POE and PEG blocks. Hydrolysis is homogeneous through the polymer backbone. Thus, its molecular weight remains relatively constant and mass loss shows quite sustained over the 14-week in vitro release. The similar phenomena are observed in the polydispersity index of the degrading copolymers. SDS-PAGE of the encapsulated BSA within the POE-PEG(5%)-POE microspheres displays that the structural integrity of BSA is intact for at least 8 weeks due to a mild environment provided by the copolymer. In addition, XPS and FTIR are utilized to investigate protein behaviors in the degrading microspheres. Protein release from the POE-PEG-POE microspheres shows a biphasic pattern, characterized by an initial stage followed by a non-detectable release. The non-release phase is dominated by either slow polymer degradation or dense microsphere matrix structures. The microsphere formulation is optimized and a sustained protein release over 2 weeks is achieved by using POE-PEG(20%)-POE at a high protein loading.     

Cyclic nucleotide-dependent protein kinases.

The actions of several hormones and neurotransmitters evoke signal transduction pathways which rapidly elevate the cytosolic concentrations of the intracellular messengers, cAMP and cGMP. The cyclic-nucleotide dependent protein kinases, cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), are the major intracellular receptors of cAMP and cGMP. These enzymes become active upon binding respective cyclic nucleotides and modulate a diverse array of biochemical events through the phosphorylation of specific substrate proteins. The focus of this review is to describe the progress made in understanding the structure and function of both PKA and PKG.     

Human erythrocyte protein 4.1 is a phosphatidylserine binding protein.

The aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS) are the major phospholipids contained in the cytoplasmic leaflet of the human erythrocyte (RBC) plasma membrane and are largely confined to that leaflet over the entire RBC lifespan. In particular, PS, which comprises approximately 13% of total RBC membrane phospholipids, is normally restricted entirely to the cytoplasmic leaflet. However, molecular mechanisms that regulate this asymmetric distribution of phospholipids are largely unknown. We examined elliptocytic RBCs that completely lacked protein 4.1 (HE [4.1 degrees]), but contained normal amounts of all other peripheral membrane proteins, and found approximately 10% of total membrane PS was accessible in the exoplasmic leaflet of these membranes. Inside out vesicles (IOVs) derived from HE [4.1 degrees] RBCs bound fewer PS liposomes than did IOVs derived from normal RBCs. Normal IOVs that were depleted of proteins 2.1 (ankyrin), 4.1, and 4.2 bound fewer PS liposomes similar to HE [4.1 degrees] IOVs, and repletion with protein 4.1 restored PS liposome binding to control levels. Addition of purified protein 4.1 to PS liposomes resulted in saturable binding with the extent of binding being proportional to the liposome PS content. Our data suggests that human RBC protein 4.1 is a PS binding protein and may be involved in the molecular mechanisms that stabilize PS in the cytoplasmic leaflet of the human RBC plasma membrane.     

High sensitivity C-reactive protein.

Though there is no apparent relationship between CRP and cardiovascular disease, the hsCRP may be the most effective single marker of disease among otherwise healthy adults, and is certainly an effective marker when employed in combination with other proven markers such as fibrinogen, homocysteine, and lipid assays. The hsCRP test may not be particularly useful among inpatients, however, as it is typically elevated in acute inflammatory disease. In addition to its apparent ability to predict the risk of cardiovascular disease, the hsCRP test points the way to further identification of risk markers.     

Interactions of surfactant protein D with bacterial lipopolysaccharides. Surfactant protein D is an Escherichia coli-binding protein in bronchoalveolar lavage.

Surfactant protein D (SP-D) is a collagenous glycoprotein that is secreted into the pulmonary airspaces by alveolar type II and nonciliated bronchiolar cells. SP-D exhibits Ca(++)-dependent carbohydrate binding in vitro and is structurally related to the collagenous C-type lectins, including serum conglutinin, serum mannose-binding proteins, and surfactant protein A. Preliminary studies showed calcium- and saccharide-dependent binding of fluorescein-conjugated or radioiodinated SP-D to a variety of microorganisms, including Gram-negative bacteria and fungi. A laboratory strain of Escherichia coli (Y1088) was chosen to further examine the mechanism(s) of binding. Binding of SP-D to Y1088 was time dependent, saturable, and inhibited by cold SP-D or competing saccharides; Scatchard analysis gave a Kd of 2 x 10(-11) M. At higher concentrations, SP-D also caused Ca(++)-dependent agglutination of Y1088 that was inhibited by alpha-glucosyl-containing saccharides, antisera to the carbohydrate-binding domain of SP-D, or Y1088 LPS. Lectin blots showed specific binding of 125I-SP-D to Y1088 LPS, as well as LPS from other several strains of enteric Gram-negative bacteria. Immunogold studies demonstrated strong and uniform surface labeling of the bacteria. Rat and human bronchoalveolar lavage (BAL) caused Ca(++)-dependent agglutination of E. coli that was dose dependent and inhibited by competing saccharides or anti-SP-D. SP-D was selectively and efficiently adsorbed from rat BAL by incubation with E. coli, and incubation of E. coli with radiolabeled rat type II cell medium revealed that SP-D is the major E. coli-binding protein secreted by freshly isolated cells in culture. We suggest that SP-D plays important roles in the lung's defense against Gram-negative bacteria.     

The role of visceral protein markers in protein calorie malnutrition.

Despite substantial evidence of the crucial role protein calorie malnutrition (PCM) plays in the occurrence of complications, increased length of stay, and cost of care in hospitalized populations, no standard approach for screening and monitoring the nutritional status of patients initially and throughout admission currently exists. Recognizing that there is a growing public and professional recognition of the importance of malnutrition, a large patient population (30-55%) at risk for PCM, and an even larger population experiencing declining nutritional status during hospitalization, this study examined the feasibility of a full-scale study to assess the value of two biochemical markers, transthyretin and albumin, for detecting and monitoring PCM in hospitalized patients. It was demonstrated that these two markers do provide important information predictive of outcomes for those they identify at risk for PCM. The patients who entered the study with or developed low transthyretin and albumin experienced poorer health outcomes and higher costs of care. Their discharge occurred in an early phase of recovery, with significant implications for after-discharge care. The full-scale study must consider severity of illness and other confounders during randomization and, preferably, be conducted in institutions that currently do not use transthyretin for nutrition assessment.     

Correcting temperature-sensitive protein folding defects.

Recently, we found that different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in correcting the temperature-sensitive protein folding defect associated with the deltaF508 cystic fibrosis transmembrane regulator (CFTR) protein. Here we examined whether the folding of other proteins which exhibit temperature-sensitive folding defects also could be corrected via a similar strategy. Cell lines expressing temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene protein pp60src, or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5 degrees C) in the presence of glycerol, trimethylamine N-oxide or deuterated water. In each case, the cells exhibited phenotypes similar to those observed when the cells were incubated at the permissive temperature (32.5 degrees C), indicative that the particular protein folding defect had been corrected. These observations, coupled with our earlier work and much older studies in yeast and bacteria, indicate that protein stabilizing agents are effective in vivo for correcting protein folding abnormalities. We suggest that this type of approach may prove to be useful for correcting certain protein folding abnormalities associated with human diseases.     

High-molecular weight kininogen. A secreted platelet protein.

Human platelets were studied immunochemically to determine if they contain high-molecular weight kininogen. On crossed immunoelectrophoresis with total kininogen antisera (antisera that recognizes both high- and low-molecular weight kininogen) extracts of platelets contained total kininogen antigen. Platelet total kininogen antigen showed complete antigenic identity with plasma total kininogen and displayed the same electrophoretic migration as plasma total kininogen. Using antisera monospecific to high molecular weight kininogen, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to directly measure platelet high-molecular weight kininogen. By CELISA, 27-101 ng of high molecular weight kininogen antigen per 10(8) platelets was quantitated in detergent-soluble lysates of washed human platelets from nine normal donors with a mean level of 60 ng +/- 24/10(8) platelets. Plasma high-molecular weight kininogen, either in the platelet suspending medium or on the surface of the platelets, could only account for 5% of antigen measured in the solubilized platelets. On the CELISA, platelet high-molecular weight kininogen was immunochemically identical to plasma and purified high-molecular weight kininogen. Platelet high-molecular weight kininogen was secreted from platelets after exposure to ionophore A23187 (3-15 microM), collagen (5-150 micrograms/ml), and thrombin (1.6 U/ml). Secreted platelet high-molecular weight kininogen did not become a part of the platelet Triton-insoluble cytoskeleton. On cross immunoelectrophoresis secreted platelet total kininogen antigen had a similar electrophoretic migration to plasma total kininogen. Thus, human platelets contain high-molecular weight kininogen that can be secreted from platelets and that may participate in plasma coagulation reactions.     

Plasma protein homocysteinylation in uremia.

Protein homocysteinylation is proposed as one of the mechanisms of homocysteine toxicity. It occurs through various means, such as the post-biosynthetic acylation of free amino groups (protein-N-homocysteinylation, mediated by homocysteine thiolactone) and the formation of a covalent -S-S- bond found primarily with cysteine residues (protein-S-homocysteinylation). Both protein modifications are a cause of protein functional derangements. Hemodialysis patients in the majority of cases are hyperhomocysteinemic, if not malnourished. Protein-N-homocysteinylation and protein-S-homocysteinylation are significantly increased in hemodialysis patients compared to controls. Oral folate treatment normalizes protein-N-homocysteinylation levels, while protein-S-homocysteinylation is significantly reduced. Albumin binding experiments after in vitro homocysteinylation show that homocysteinylated albumin is significantly altered at the diazepam, but not at the warfarin and salicilic acid binding sites.     

Influence of dietary protein intake on whole-body protein turnover in humans.

Methods for measuring rates of protein synthesis and degradation in the whole body of humans with isotopes of carbon and nitrogen are described and attention is drawn to their relative merits and drawbacks for studying the nutritional control of protein metabolism. A review of published work on dietary protein and protein metabolism leads to the conclusion that protein is the major dietary determinant of whole-body protein turnover rates, and that energy intake is comparatively unimportant. Dietary protein affects protein turnover at two levels: an immediate response to the intake of protein in meals and a longer-term adaptation after a change in protein intake. An increase in the level of dietary protein enhances the response to meals, which mainly consists of a decrease in the rate of protein degradation. The adaptation to higher protein intakes involves an increase in the basal (postabsorptive) rates of both synthesis and degradation. Suggestions for future investigation include more detailed studies of the acute and adaptive responses, to facilitate understanding of dietary protein requirements, and the effects of very-high-protein intakes with continued development of techniques for studying protein turnover in individual tissues in humans.     

Absorption enhancers in pulmonary protein delivery.

Extensive research efforts have been directed towards the systemic administration of therapeutic proteins and poorly absorbed macromolecules via various nontraditional, injection-free administration sites such as the lung. As a portal for noninvasive delivery, pulmonary administration possesses several attractive features including a large surface area for drug absorption. Nevertheless, achieving substantial bioavailability of proteins and macromolecules by this route has remained a challenge, chiefly due to poor absorption across the epithelium. The lungs are relatively impermeable to most drugs when formulated without an absorption enhancer/promoter. In an attempt to circumvent this problem, many novel absorption promoters have been tested for enhancing the systemic availability of drugs from the lungs. Various protease inhibitors, surfactants, lipids, polymers and agents from other classes have been tested for their efficacy in improving the systemic availability of protein and macromolecular drugs after pulmonary administration. The purpose of this article is to provide the reader with a summary of recent advances made in the field of pulmonary protein delivery utilizing absorption enhancers. This report reviews the various agents used to increase the bioavailability of these drugs from the lungs, their mechanisms of action and effectiveness, and their potential for toxicity.