Mikhail I. Shakhparonov

Diminished NADPH transhydrogenase activity and mitochondrial redox regulation in human failing myocardium

Although the functional role of nicotinamide nucleotide transhydrogenase (Nnt) remains to be fully elucidated, there is strong evidence that Nnt plays a critical part in mitochondrial metabolism by maintaining a high NADPH-dependent GSH/GSSG ratio, and thus the control of cellular oxidative stress. Using real-time PCR, spectrophotometric and western blotting techniques, we sought to determine the presence, abundance and activity level of Nnt in human heart tissues and to discern whether these are altered in chronic severe heart failure. Left ventricular levels of the NNT gene and protein expression did not differ significantly between the non-failing donor (NF) and heart failure (HF) group. Notably, compared to NF, Nnt activity rates in the HF group were 18% lower, which coincided with significantly higher levels of oxidized glutathione, lower glutathione reductase activity, lower NADPH and a lower GSH/GSSG ratio. In the failing human heart a partial loss of Nnt activity adversely impacts NADPH-dependent enzymes and the capacity to maintain membrane potential, thus contributing to a decline in bioenergetic capacity, redox regulation and antioxidant defense, exacerbating oxidative damage to cellular proteins.

Identification of a novel gene of the X,K-ATPase β-subunit family that is predominantly expressed in skeletal and heart muscles1

We have identified the fifth member of the mammalian X,K‐ATPase β‐subunit gene family. The human and rat genes are largely expressed in skeletal muscle and at a lower level in heart. The deduced human and rat proteins designated as βmuscle (βm) consist of 357 and 356 amino acid residues, respectively, and exhibit 89% identity. The sequence homology of βm proteins with known Na,K‐ and H,K‐ATPase β‐subunits are 30.5–39.4%. Unlike other β‐subunits, putative βm proteins have large N‐terminal cytoplasmic domains containing long Glu‐rich sequences. The data obtained indicate the existence of hitherto unknown X,K‐ATPase (most probably Na,K‐ATPase) isozymes in muscle cells.

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic α subunit1

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic α subunit of a non‐gastric ouabain‐sensitive H,K‐ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT‐PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue‐specific expression patterns indicates that animal and human non‐gastric H,K‐ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non‐gastric H,K‐ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).

Proteome–Metabolome Profiling of Ovarian Cancer Ascites Reveals Novel Components Involved in Intercellular Communication

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell–cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.

Detection of Transglutaminase 2 conformational changes in living cell.

Transglutaminase 2 (TG2) is a ubiquitous Ca(2+)-dependent protein cross-linking enzyme that is implicated in a variety of biological disorders. In in vitro experiments when Ca(2+) concentration was increased TG2 changed its conformation and was able to cross-link other proteins via formation of an isopeptide bond. However the mechanisms that regulate TG2 transamidation activity in cells are still unknown. In this study we have developed FRET-based method for monitoring TG2 conformation changes and, probably, cross-linking activity in living cells. Using this approach we have showed that a significant amount of TG2 within the cell is accumulated in perinuclear endosomes and has a cross-linking inactive conformation, while TG2 that is located beneath the cell membrane has a transamidation active conformation. After the induction of apoptosis cytoplasmic TG2 changed its conformation and activates while, TG2 in endosomes retained transamidation inactive conformation even at late stages of apoptosis.

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase.

Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

Nuclear transport of protein TTC4 depends on the cell cycle

TTC4 (tetratricopeptide repeat domain protein 4) is a putative tumor suppressor involved in the transformation of melanocytes. At present, the relationships between TTC4 and DNA replication proteins are largely unknown, as are the tissue distribution and subcellular localization of TTC4. Using reverse transcription with the polymerase chain reaction, we have observed that the murine TTC4 gene is ubiquitously expressed. Analysis of the TTC4 subcellular localization has shown that, upon overexpression, TTC4 localizes to the cytoplasm. Interestingly, co-expression with a known protein interaction partner, hampin/MSL1, results in the nuclear translocation of the TTC4 protein. The subcellular localization of endogenous TTC4 depends, however, on the cell cycle: it is mostly nuclear in the G1 and S phases and is evenly distributed between the nucleus and cytoplasm in G2. The nuclear transport of TTC4 is apparently a complex process dependent on interactions with other proteins during the progression of the cell cycle. Thus, the dynamic character of the nuclear accumulation of TTC4 might be a potential link with regard to its function in tumor suppression.

Decreased expression of the human immunoglobulin J-chain gene in squamous cell cancer and adenocarcinoma of the lungs

Secreted polymeric immunoglobulins, IgA dimers and IgM pentamers, are unique in having not only the L and H chains but also the J chain, which is responsible for oligomerization. These antibodies belong to the local adaptive immune system, located in the mucosas of the respiratory and digestive systems and acting first to protect the body from infectious agents. Polymeric immunoglobulins are produced by highly specific B cells and are actively transported to the mucosa surface through epithelial cells. Hence, their production and, accordingly, the J-chain content are associated with the transporting function of the epithelium and depend on its state, which dramatically changes upon malignant transformation. The content of the J chain and the transcription level of its gene were studied in normal and tumor cells at various stages of squamous cell cancer and adenocarcinoma of the lungs by RT-PCR and immunoblotting.

The effect of ablation of the gene for H+-transporting NAD/NADP transhydrogenase on the life spans of nematodes and mammals

Mitochondrial transhydrogenase catalyzes the reaction; Hout+ + NADP+ + NADH = NAD+ + NADPH + Hin+. The maintenance of the NADPH pool increases the mitochondrial antioxidant potential. Therefore, according to the commonly adopted free radical theory of aging, ablation of the transhydrogenase gene should reduce the life span. However, contrary to this reasoning, the life span of Caenorhabditis elegans nematodes with null mutations in the gene does not differ from that in wild-type worms. This fact indicates that free radical damage of mitochondria is not associated with aging. Meta analysis of data on the life span in mice possessing a spontaneous mutation in the transhydrogenase gene shows that a lack of this enzyme does not accelerate aging in mammals either. The heart is the tissue with the highest transhydrogenase production rate, and it is likely that this enzyme contributes to the protection of cardiac myocytes from oxidative stress.

Nongastric H,K-ATPase: structure and functional properties.

Abstract: Nongastric H,K‐ATPases whose catalytic subunits (AL1) encoded by human ATP1AL1 and homologous animal genes comprise the third distinct group within the X,K‐ATPase family. No unique nongastric β has been identified. Precise in situ colocalization and strong association of AL1 with β1 of Na,K‐ATPase was detected in apical membranes of rodent prostate epithelium. In this tissue, β1NK serves as an authentic subunit of both the Na,K‐ and nongastric H,K‐pumps. Upon expression in Xenopus oocytes the human AL1 can assemble with β1NK, and more efficiently with gastric βHK, into functional H,K‐pumps. Both AL1/β complexes exhibit a similar K‐affinity, and their K‐transport depends on intra‐ and extracellular Na. These data provide new evidence that nongastric H,K‐ATPase can perform Na/K‐exchange, and indicate that β does not significantly affect this ion‐pump function. Analysis of human nongastric H,K‐ATPase expressed in Sf‐21 insect cells revealed that AL1/βHK exhibits substantial enzymatic activities in K‐free medium and K stimulates, but Na has inhibitory effect on ATP hydrolysis. Thus, although the nongastric H,K‐ATPase can function as Na/K exchanger, its reaction mechanism is different from that of the Na,K‐ATPase. Human nongastric H,K‐ATPase is highly sensitive to bufalin, digoxin, and digitoxin, but almost resistant to digoxigenin and ouabagenin.