Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

DPS-Like Peroxide Resistance Protein: Structural and Functional Studies on a Versatile Nanocontainer

Oxidative stress is a constant threat to almost all organisms. It damages a number of biomolecules and leads to the disruption of many crucial cellular functions. It is caused by reactive oxygen species (ROS), such as hydrogen peroxide (H2O₂), superoxide (•O₂ -), and hydroxyl radical (•OH). The most harmful of these compounds is •OH, which is only formed in cells in the presence of redox-cycling transition metals, such as iron and copper. Bacteria have developed a number of mechanisms to cope with ROS. One of the most widespread means employed by bacteria is the DNA-binding proteins from starved cells (Dps). Dps proteins protect the cells by binding and oxidizing Fe2+, thus greatly reducing the production of •OH. The oxidized iron is stored inside the protein as an iron core. In addition, Dps proteins bind directly to DNA forming a protective coating that shields DNA from harmful agents. Moreover, Dps proteins have been found to elicit other protective functions in cells and to participate in bacterial virulence. Dps proteins are of special importance to Streptococci owing to the lack of catalase in this genus of bacteria.This study was focused on structural and functional characterization of streptococcal Dpslike peroxide resistance (Dpr) proteins. Initially, crystal structures of Streptococcus pyogenes Dpr were determined. The data confirmed the presence of a di-metal ferroxidase center (FOC) in Dpr proteins and revealed the presence of a novel N-terminal helix as well as a surface metal-binding site. The crystal structures of Streptococcus suis Dpr complexed with transition metals demonstrated the metal specificity of the FOC. Solution binding studies also indicated the presence of a di-metal FOC. These results suggested a possible role for Dpr in the detoxification of various metals. Iron was found to mineralize inside the protein as ferrihydrite based on X-ray absorption spectroscopy data. The iron core was found to exhibit clear superparamagnetic behaviour using magnetic and Mössbauer measurements. The results from this study are expected to further increase our understanding on the binding, oxidation, and mineralization of iron and other metals in Dpr proteins. In particular, the structural and magnetic properties of the iron core can form a basis for potential new applications in nanotechnology. From the streptococcal viewpoint, the results would help in understanding better the complicated picture of bacterial pathogenesis. Dpr proteins may also provide a novel target for drug design due to their tight involvement in bacterial virulence.

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

Novel Functions of ErbB4 and NRG-1 in Development and Cancer

Cells communicate, or signal, with each other constantly to ensure proper functioning of tissues and organs. Cell signaling is often performed by interplay of receptors and ligands that bind these receptors. ErbB receptors (epidermal growth factor receptors, EGFR, HER) bind extracellular growth factors and transduce these signals inside of cells. ErbB dysfunction promotes carcinogenesis, and also results in numerous defects during normal development. This study focused on the functions of one member of the ErbB receptor family, ErbB4, and growth factor, neuregulin-1 (NRG-1), that can bind and activate ErbB4. This study aimed to find novel functions of ErbB4 and NRG-1. Hypoxia, or deficiency of oxygen, is common in cancer and ischemic conditions. One of the key findings of the work was the identification and characterization of a cross-talk between ErbB4 and Hypoxia-inducible factor 1α (HIF-1α), the central mediator of hypoxia signaling. ErbB4 activation by NRG-1 was found to increase HIF-1α activity. Interestingly, this regulation occurred in reciprocal manner as HIF-1α was also able to increase protein levels of NRG-1 and ErbB4. Moreover, expression of NRG-1 and ErbB4 was associated with HIF activity in vivo in human clinical samples and in mice. Reduction of functional ErbB4 in developing zebrafish embryos resulted in defects in development of the skeletal muscles. To study ErbB4 functions in pathological situation in humans, clinical samples of serous ovarian carcinoma were analyzed using tissue microarrays and real-time RT-PCR. A specific isoform of ErbB4, CYT-1, was associated with poor survival in serous ovarian cancer and increased anchorage independent growth of ovarian cancer cells in vitro. These observations demonstrate that ErbB4 and NRG-1 are essential regulators of cellular response to hypoxia, of development, and of ovarian carcinogenesis.

Regulation of gap junctions by protein phosphorylation

Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

Functions of the extracellular matrix and matrix degrading proteases during tumor progression

Cell interactions with extracellular matrices are important to pathological changes that occur during cell transformation and tumorigenesis. Several extracellular matrix proteins including fibronectin, thrombospondin-1, laminin, SPARC, and osteopontin have been suggested to modulate tumor phenotype by affecting cell migration, survival, or angiogenesis. Likewise, proteases including the matrix metalloproteinases (MMPs) are understood to not only facilitate migration of cells by degradation of matrices, but also to affect tumor formation and growth. We have recently demonstrated an in vivo role for the RGD-containing protein, osteopontin, during tumor progression, and found evidence for distinct functions in the host versus the tumor cells. Because of the compartmentalization and temporal regulation of MMP expression, it is likely that MMPs may also function dually in host stroma and the tumor cell. In addition, an important function of proteases appears to be not only degradation, but also cleavage of matrix proteins to generate functionally distinct fragments based on receptor binding, biological activity, or regulation of growth factors.

Protein folding: a perspective for biology, medicine and biotechnology

At the present time, protein folding is an extremely active field of research including aspects of biology, chemistry, biochemistry, computer science and physics. The fundamental principles have practical applications in the exploitation of the advances in genome research, in the understanding of different pathologies and in the design of novel proteins with special functions. Although the detailed mechanisms of folding are not completely known, significant advances have been made in the understanding of this complex process through both experimental and theoretical approaches. In this review, the evolution of concepts from Anfinsen's postulate to the "new view" emphasizing the concept of the energy landscape of folding is presented. The main rules of protein folding have been established from in vitro experiments. It has been long accepted that the in vitro refolding process is a good model for understanding the mechanisms by which a nascent polypeptide chain reaches its native conformation in the cellular environment. Indeed, many denatured proteins, even those whose disulfide bridges have been disrupted, are able to refold spontaneously. Although this assumption was challenged by the discovery of molecular chaperones, from the amount of both structural and functional information now available, it has been clearly established that the main rules of protein folding deduced from in vitro experiments are also valid in the cellular environment. This modern view of protein folding permits a better understanding of the aggregation processes that play a role in several pathologies, including those induced by prions and Alzheimer's disease. Drug design and de novo protein design with the aim of creating proteins with novel functions by application of protein folding rules are making significant progress and offer perspectives for practical applications in the development of pharmaceuticals and medical diagnostics.

Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18) and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA) by mouse peritoneal macrophages. We observed that a) macrophages are able to recognize (bind to) these red cells, b) this interaction can be inhibited by denatured BSA in the fluid phase, c) there is no phagocytosis of these particles by normal macrophages, d) phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e) this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A).

Role of Tamm-Horsfall protein and uromodulin in calcium oxalate crystallization

One of the defenses against nephrolithiasis is provided by macromolecules that modulate the nucleation, growth, aggregation and retention of crystals in the kidneys. The aim of the present study was to determine the behavior of two of these proteins, Tamm-Horsfall and uromodulin, in calcium oxalate crystallization in vitro. We studied a group of 10 male stone formers who had formed at least one kidney stone composed of calcium oxalate. They were classified as having idiopathic nephrolithiasis and had no well-known metabolic risk factors involved in kidney stone pathogenesis. Ten normal men were used as controls, as was a group consisting of five normal women and another consisting of five pregnant women. Crystallization was induced by a fixed supersaturation of calcium oxalate and measured with a Coulter Counter. All findings were confirmed by light and scanning electron microscopy. The number of particulate material deposited from patients with Tamm-Horsfall protein was higher than that of the controls (P<0.001). However, Tamm-Horsfall protein decreased the particle diameter of the stone formers when analyzed by the mode of the volume distribution curve (P<0.002) (5.64 ± 0.55 µm compared to 11.41 ± 0.48 µm of uromodulin; 15.94 ± 3.93 µm and 12.45 ± 0.97 µm of normal men Tamm-Horsfall protein and uromodulin, respectively; 8.17 ± 1.57 µm and 9.82 ± 0.95 µm of normal women Tamm-Horsfall protein and uromodulin, respectively; 12.17 ± 1.41 µm and 12.99 ± 0.51 µm of pregnant Tamm-Horsfall protein and uromodulin, respectively). Uromodulin produced fewer particles than Tamm-Horsfall protein in all groups. Nonetheless, the total volume of the crystals produced by uromodulin was higher than that produced by Tamm-Horsfall protein. Our results indicate a different effect of Tamm-Horsfall protein and uromodulin. This dual behavior suggests different functions. Tamm-Horsfall protein may act on nucleation and inhibit crystal aggregation, while uromodulin may promote aggregation of calcium oxalate crystals.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

High doses of riboflavin and the elimination of dietary red meat promote the recovery of some motor functions in Parkinson's disease patients

Abnormal riboflavin status in the absence of a dietary deficiency was detected in 31 consecutive outpatients with Parkinson's disease (PD), while the classical determinants of homocysteine levels (B6, folic acid, and B12) were usually within normal limits. In contrast, only 3 of 10 consecutive outpatients with dementia without previous stroke had abnormal riboflavin status. The data for 12 patients who did not complete 6 months of therapy or did not comply with the proposed treatment paradigm were excluded from analysis. Nineteen PD patients (8 males and 11 females, mean age ± SD = 66.2 ± 8.6 years; 3, 3, 2, 5, and 6 patients in Hoehn and Yahr stages I to V) received riboflavin orally (30 mg every 8 h) plus their usual symptomatic medications and all red meat was eliminated from their diet. After 1 month the riboflavin status of the patients was normalized from 106.4 ± 34.9 to 179.2 ± 23 ng/ml (N = 9). Motor capacity was measured by a modification of the scoring system of Hoehn and Yahr, which reports motor capacity as percent. All 19 patients who completed 6 months of treatment showed improved motor capacity during the first three months and most reached a plateau while 5/19 continued to improve in the 3- to 6-month interval. Their average motor capacity increased from 44 to 71% after 6 months, increasing significantly every month compared with their own pretreatment status (P < 0.001, Wilcoxon signed rank test). Discontinuation of riboflavin for several days did not impair motor capacity and yellowish urine was the only side effect observed. The data show that the proposed treatment improves the clinical condition of PD patients. Riboflavin-sensitive mechanisms involved in PD may include glutathione depletion, cumulative mitochondrial DNA mutations, disturbed mitochondrial protein complexes, and abnormal iron metabolism. More studies are required to identify the mechanisms involved.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54% according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 ± 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 ± 25.9 and 61.18 ± 25.04 mL min-1 (1.73 m²)-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2%) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7%) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6%) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca2+ signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.