Zfra is a 31-amino-acid zinc finger-like protein, which participates in the tumor necrosis factor signaling. Here, we determined that when nude mice and BALB/c mice were pre-injected with nanogram levels of a synthetic Zfra1–31 or truncated Zfra4–10 peptide via tail veins, these mice became resistant to the growth, metastasis and stemness of melanoma cells, and many malignant cancer cells. The synthetic peptides underwent self-polymerization in phosphate-buffered saline. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra aggregation and its-mediated cancer suppression in vivo. Injected Zfra peptide autofluoresced due to polymerization and was trapped mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naïve mice conferred resistance to cancer growth. Zfra-binding cells, designated Hyal-2+ CD3− CD19− Z cells, are approximately 25–30% in the normal spleen, but are significantly downregulated (near 0–3%) in tumor-growing mice. Zfra prevented the loss of Z cells caused by tumors. In vitro stimulation or education of naïve spleen cells with Zfra allowed generation of activated Z cells to confer a memory anticancer response in naïve or cancer-growing mice. In particular, Z cells are abundant in nude and NOD-SCID mice, and can be readily activated by Zfra to mount against cancer growth.

The red blood cell or erythrocyte is easily purified, readily available, and has a relatively simple structure. Therefore, it has become a very well studied cell in terms of protein composition and function. RBC proteomic studies performed over the last five years, by several laboratories, have identified 751 proteins within the human erythrocyte. As RBCs contain few internal structures, the proteome will contain far fewer proteins than nucleated cells. In this minireview, we summarize the current knowledge of the RBC proteome, discuss alterations in this partial proteome in varied human disease states, and demonstrate how in silico studies of the RBC interactome can lead to considerable insight into disease diagnosis, severity, and drug or gene therapy response. To make these latter points we focus on what is known concerning changes in the RBC proteome in Sickle Cell Disease.

Actin oxidation is known to result in changes in cytoskeleton organization and dynamics. Actin oxidation is clinically relevant since it occurs in the erythrocytes of sickle cell patients and may be the direct cause of the lack of morphological plasticity observed in irreversibly sickled red blood cells (ISCs). During episodes of crisis, ISCs accumulate C284-C373 intra-molecularly disulfide bonded actin, which reduces actin filament dynamics. Actin cysteines 284 and 373 (285 and 374 in yeast) are conserved, suggesting that they play an important functional role. We have been investigating the physiological roles of these cysteines using the model eukaryote S. cerevisiae in response to oxidative stress load. During acute oxidative stress, all of the F-actin in wild type cells collapses into a few puncta that we call oxidized actin bodies (OABs). In contrast, during acute oxidative stress the actin cytoskeleton in Cys-to-Ala actin mutants remains polarized longer, OABs are slower to form, and the cells recover more slowly than wild type cells, suggesting that the OABs play a protective role. Live cell imaging revealed that OABs are large, immobile structures that contain actin binding proteins and that can form by the fusion of actin cortical patches. We propose that actin’s C285 and C374 may help to protect the cell from oxidative stress arising from normal oxidative metabolism and contribute to the cell’s general adaptive response to oxidative stress.

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined. Molecular & Cellular Proteomics 3:501-509, 2004.A human red blood cell (RBC) 1 is in residence in the human circulatory system for 120 days carrying oxygen from the lungs to all tissues within the body and carbon dioxide from the tissues back to the lungs. An RBC is an 8-m biconcave disk bounded by a plasma membrane. The major cytoplasmic constituent is hemoglobin, which is responsible for binding and releasing oxygen and carbon dioxide. On the cytoplasmic surface of the plasma membrane is a two-dimensional meshwork of proteins referred to as spectrin membrane skeleton. The spectrin membrane skeleton renders elasticity and flexibility to an RBC, allowing it to pass through vessels and capillaries that narrow to 1 m in diameter (1).Because of the ease in obtaining RBCs and because they lack internal organelles, the plasma membrane of this cell type has been studied extensively. The functions of hemoglobin are also well documented. Based on four decades of study, the identity, function, and topology of many RBC membrane proteins have been determined (1-3). With the advent of modern mass spectrometry (MS) and associated proteomic techniques, determination of the RBC proteome is now plausible. This kind of approach is a necessary first step in understanding how the RBC proteome becomes altered in various hematologic disorders. With this goal in mind, we utilized ion trap ...

In this minireview, we focus on advances in our knowledge of the human erythrocyte proteome and interactome that have occurred since our seminal review on the topic published in 2007. As will be explained, the number of unique proteins has grown from 751 in 2007 to 2289 as of today. We describe how proteomics and interactomics tools have been used to probe critical protein changes in disorders impacting the blood. The primary example used is the work done on sickle cell disease where biomarkers of severity have been identified, protein changes in the erythrocyte membranes identified, pharmacoproteomic impact of hydroxyurea studied and interactomics used to identify erythrocyte protein changes that are predicted to have the greatest impact on protein interaction networks.

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.

The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles. A two-dimensional differential in-gel electrophoresis (2D-DIGE) approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs. Twenty-eight proteins were identified with altered protein content in response to lactacystin. Seven cytosolic proteins showed an increase and 16 showed a decrease; five membrane proteins showed a decrease. We conclude that the proteins showing increased abundance are either primary or secondary targets of the 20S proteasome and that putatively degraded proteins are secondary targets. Therefore, functional 20S proteasomes exist within mature RBCs. Our study did not detect 26S proteasome activity using the 2D-DIGE approach.

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys The mitochondrial citrate transport protein (CTP) 1 from higher eukaryotes catalyzes an obligatory exchange of citrate plus a proton across the mitochondrial inner membrane for either another tricarboxylate ϩ H ϩ , a dicarboxylate, or phosphoenolpyruvate (1). The CTP plays an essential role in intermediary metabolism in that it supplies the cytoplasm with citrate, which can then function as a carbon source for fatty acid and sterol biosyntheses and can also generate NAD ϩ for use in glycolysis (2-5). Because of its importance, the CTP has been extensively characterized. Thus, it has been purified and reconstituted (6, 7), kinetically characterized (8), cloned (9), and overexpressed (10). More recently, the mitochondrial CTP from the yeast Saccharomyces cerevisiae has been identified via overexpression followed by functional reconstitution of the purified protein product (11).In the present paper we report the construction and optimization of a cysteine-less (i.e. Cys-less) yeast mitochondrial CTP, which upon overexpression and functional reconstitution displays properties that are quite similar to the wild-type transporter. We also report novel effects of sulfhydryl reagents on the functioning of single, double, and triple Cys replacement mutants that were sequentially constructed during the development of the final Cys-less CTP. Finally, we report the use of the Cys-less CTP to probe the roles of Arg 181 and Arg 189 in the transport mechanism. This work not only demonstrates that cysteines are not essential to the CTP translocation mechanism but importantly: (i) provides insight as to which cysteines are responsible for sulfhydryl reagent-mediated inhibition; (ii) demonstrates the suitability of the Cys-less CTP for a variety of structure/function analyses; and (iii) provides important new information regarding the role of positive charge and Cys 192 within transmembrane domain IV of the CTP. EXPERIMENTAL PROCEDURESSite-directed Mutagenesis, Overexpression, and Isolation of the Citrate Transport Protein Mutants-Mutant CTP genes were prepared utilizing the PCR Site-Directed Mutagenesis System (Life Technologies, Inc.). This system combines the use of PCR to introduce the desired mutation followed by uracil DNA glycosylase cloning. The PCR amplifications and the subsequent cloning steps were carried out according to the manufacturer's instructions. Mutagenic primers were designed to yield the following codon replacements. All wild-type cysteines (i.e. residues 28, 73, 192, and 256) were encoded by TGT and were replaced with the TCT codon for Ser. In later experiments, the Ser 73 codon (TCT) was further mutated to the Val codon (GTT). The Arg 1...