Cellular regulation and molecular interactions of the ferritins

Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature’s unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism. Received 25 June 2005; received after revision 17 October 2005; accepted 25 November 2005     

Etiologic factors in Paget’s disease of bone

Paget’s disease of bone is a chronic focal skeletal disorder characterized by increased bone resorption by the osteoclasts. Paramyxoviral gene products have been detected in pagetic osteoclasts. Paget’s disease is an autosomal dominant trait with genetic heterogeneity. Several mutations in the ubiquitin-associated (UBA) domain of sequestosome 1 (SQSTM1/p62) have been identified in patients with Paget’s disease. Similarly, mutations in the valosin-containing protein (VCP) gene have been shown to cause inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia. In addition, gene polymorphisms and enhanced levels of cytokine/growth factors associated with Paget’s disease have been identified. However, the etiologic factors in Paget’s disease remain elusive. A cause and effect relationship for the paramyxoviral infection and SQSTM1/ p62 gene mutations responsible for pagetic osteoclast development and disease severity are unclear. This article will highlight the etiologic factors involved in the pathogenesis of Paget’s disease. Received 6 October 2005; received after revision 2 November 2005; accepted 24 November 2005     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

Crystallin proteins and amyloid fibrils

Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, α-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer’s and Parkinson’s). In this review, the literature on the interaction of αB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials. Received 13 June 2008; received after revision 29 July 2008; accepted 05 August 2008     

Cytotoxic activity of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide is underlain by DNA interchain cross-linking

Currently, chemical bifunctional cross-linkers are regarded as promising therapeutic agents capable of affecting cell metabolism. Depending on the nature of the active groups and on the length of their mediating spacer, these cross-linkers have been shown to influence mitochondrial functions, the cell cycle and cell death. The current study was aimed to assay cellular effects of a cross-linker with ‘zero’-length spacer, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). When added to cultures of transformed cells, EDC induced a G2/M blockade followed by cell death. Analysis of the molecular targets revealed that alteration of the cell cycle was caused by EDC-induced interchain cross-linking within double-stranded DNA. Administration of EDC to animals with experimental tumors increased their life span. The analysis of tumor cells from EDC-treated mice showed up-regulation of p21/WAF1, disturbance of tumor cell cytokinesis and, hence, cell death. Thus, both in vitro and in vivo, EDC exhibits cytotoxic activity, which may be of potential therapeutic use. Received 15 August 2005; received after revision 23 September 2005; accepted 15 November 2005     

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons

Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent major dementia-associated abnormalities in Alzheimer’s disease (AD). This study examined the role of oxidative stress as a factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated with H₂O₂ (9–90 μM) or desferrioxamine (2–25 μM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function, and pro-apoptosis and sprouting gene expression. H₂O₂ treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The H₂O₂-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin, and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection), and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast, hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD, both oxidative stress and hypoxic injury can contribute to AD neurodegeneration. Received 24 May 2000; received after revision 7 July 2000; accepted 27 July 2000     

Multiple roles of the DSCR1 (Adapt78 or RCAN1) gene and its protein product Calcipressin 1 (or RCAN1) in disease

The DSCR1 (Adapt78) gene¹ is transiently induced by stresses to temporarily protect cells against further potentially lethal challenges. However, chronic expression of the DSCR1 (Adapt78) gene has now been implicated in several pathological conditions including Alzheimer’s disease, Down syndrome and cardiac hypertrophy. Calcipressin 1 has been shown to function through direct binding and inhibition of the serine threonine protein phosphatase Calcineurin. Pharmacological inhibition of calcineurin, by the immunosuppressive drugs cyclosporin A and FK506, affects a wide variety of diseases. It is, therefore, likely that this endogenous calcineurin inhibitor, calcipressin 1, may also play a role in a variety of human diseases. ¹Please note that the mammalian DSCR1 gene is also called Adapt78 or RCAN1, and its protein products have been named Calcipressin1, MCIP1 and RCAN1. A proposal to adopt a single gene name of RCAN1 and a protein name RCAN1 (for Regulator of Calcineurin) has been endorsed by the HUGO Gene Nomenclature Committee, but final approval must await agreement from a majority of researchers in the field. Received 2 March 2005; received after revision 27 May 2005; accepted 19 July 2005     

Mitochondrial association of alpha-synuclein causes oxidative stress

α-Synuclein is a neuron-specific protein that contributes to the pathology of Parkinson’s disease via mitochondria-related mechanisms. The present study investigated possible interaction of α-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing α-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that α-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of α-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by α-synuclein. Received 27 December 2007; received after revision 7 February 2008; accepted 8 February 2008     

Heterogeneous bioenergetic behaviour of subsarcolemmal and intermyofibrillar mitochondria in fed and fasted rats

This study was designed to examine energetic behaviour of skeletal muscle subsarcolemmal and intermyofibrillar mitochondrial populations. The data show that subsarcolemmal mitochondria exhibited a lower degree of coupling and efficiency than intermyofibrillar ones, and can therefore be considered less efficient at producing ATP. In addition, subsarcolemmal mitochondria showed an increased sensitivity to palmitate-induced uncoupling, in line with high adenine nucleotide translocator content and decreased oxidative damage. We then determined the effect of 24 h fasting on energetic characteristics of skeletal muscle mitochondrial populations. We found that fasting enhanced proton leak and decreased the degree of coupling and efficiency, both in the absence and in the presence of palmitate only in subsarcolemmal mitochondria. Moreover, this mitochondrial population showed lower oxidative damage, probably due to a counter-regulatory mechanism mediated by uncoupling protein 3. Subsarcolemmal and intermyofibrillar mitochondria appear to exhibit different energetic characteristics and can be differently affected by physiological stimuli. Received 28 September 2005; received after revision 9 November 2005; accepted 28 November 2005     

Downregulation of NF-κB signaling by mutant huntingtin proteins induces oxidative stress and cell death

Accumulation of abnormal proteins and endoplasmic reticulum stress accompany neurodegenerative diseases including Huntington’s disease. We show that the expression of mutant huntingtin proteins with extended polyglutamine repeats differentially affected endoplasmic reticulum signaling cascades linked to the inositol-requiring enzyme-1 (IRE1) pathway. Thus, the p38 and c-Jun N-terminal kinase pathways were activated, while the levels of the nuclear factor-κB-p65 (NF-κB-p65) protein decreased. Downregulation of NF-κB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death. Concomitantly, calpain was activated, and treatment with calpain inhibitors restored NF-κB-p65 levels and increased cell viability. The calpain regulator, calpastatin, was low in cells expressing mutant huntingtin, and overexpression of calpastatin counteracted the deleterious effects caused by N-terminal mutant huntingtin proteins. These results show that calpastatin and an altered NF-κB-p65 signaling are crucial factors involved in oxidative stress and cell death mediated by mutant huntingtin proteins.     

Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain

Reggies (flotillins) are detergent-resistant microdomains involved in the scaffolding of large heteromeric complexes that signal across the plasma membrane. Based on the presence of an evolutionarily widespread motif, reggies/flotillins have been included within the SPFH (stomatin-prohibitin-flotillin-HflC/K) protein superfamily. To better understand the origin and evolution of reggie/flotillin structure and function, we searched databases for reggie/flotillin and SPFH-like proteins in organisms at the base and beyond the animal kingdom, and used the resulting dataset to compare their structural and functional domains. Our analysis shows that the SPFH grouping has little phylogenetic support, probably due to convergent evolution of its members. We also find that reggie/flotillin homologues are highly conserved among metazoans but are absent in plants, fungi and bacteria, where only proteins with ‘reggie-like’ domains can be found. However, despite their low sequence similarities, reggie/flotillin and ‘reggie-like’ domains appear to subserve related functions, suggesting that their basic biological role was acquired independently during evolution. Received 21 September 2005; received after revision 14 November 2005; accepted 21 November 2005     

Human selenoproteins at a glance

The public perception of selenium has changed significantly over the last decades. Originally mainly known for its high toxicity, it was later recognized as an essential trace element and is now (despite its narrow therapeutic window) almost being marketed as a lifestyle drug. Indeed, some clinical and preclinical studies suggest that selenium supplementation may be beneficial in a large number of clinical conditions. However, its mode of action is unresolved in most of these cases. Selenocysteine – identified as the 21^st amino acid used in ribosome-mediated protein synthesis – is incorporated in at least 25 specific, genetically determined human selenoproteins, many of which have only recently been discovered. Restoration of normal selenoprotein levels may be – apart from direct supranutritional effects – one possible explanation for the effects of selenium supplements. In this review we provide a brief but up-to-date overview of what is currently known about these 25 acknowledged human selenoproteins and their synthesis. Received 30 March 2005; received after revision 4 July 2005; accepted 13 July 2005     

Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells

Mutations in CLCN5, which encodes the voltage-dependent Cl⁻/H⁺antiporter, CLC-5, cause Dent’s disease. This disorder is characterized by low molecularweight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent’s disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis. Received 22 October 2005; received after revision 26 November 2005; accepted 2 December 2005     

Hammerhead ribozyme design and application

The emerging knowledge about RNA-based enzymes has already had great impact on our concept of evolutionary history, making the ‘RNA world’ more likely. It may well have an equally important impact on the diagnostic and therapeutic practices of human and veterinary medicine in the next decade. We are not quite there yet. This review addresses the design and application of hammerhead ribozymes, two aspects of a conserved and most commonly studied and used enzymatically active entity among the RNA enzymes. The emerging picture is one of great diversity. There is at this stage no general cell model nor a clearly preferable ribozyme structure. Each and every cell line (and tissue) may be unique in that they vary with respect to structural requirements for optimal uptake, activity and stability of ribozymes. We may have seen only the tip of the iceberg when it comes to RNA-based enzymes and their roles in biology and medicine. Received 3 June 1998; received after revision 28 July 1998; accepted 28 July 1998     

Antimicrobial skin peptides and proteins

Human skin is permanently exposed to microorganisms, but rarely infected. One reason for this natural resistance might be the existence of a ‘chemical barrier’ consisting in constitutively and inducibly produced antimicrobial peptides and proteins (AMPs). Many of these AMPs can be induced in vitro by proinflammatory cytokines or bacteria. Apart from being expressed in vivo in inflammatory lesions, some AMPs are also focally expressed in skin in the absence of inflammation. This suggests that non-inflammatory stimuli of endogenous and/or exogenous origin can also stimulate AMP synthesis without inflammation. Such mediators might be ideal ‘immune stimulants’ to induce only the innate antimicrobial skin effector molecules without causing inflammation. Received 9 August 2005; received after revision 21 October 2005; accepted 16 November 2005     

Immunophilins: for the love of proteins

Immunophilins are chaperones that may also exhibit peptidylprolyl isomerase (PPIase) activity. This review summarizes our knowledge of the two largest families of immunophilins, namely cyclophilin and FK506-binding protein, and a novel chimeric dual-family immunophilin, named FK506- and cyclosporin-binding protein (FCBP). The larger members of each family are modular in nature, consisting of multiple PPIase and/or protein-protein interaction domains. Despite the apparent difference in their sequence and three-dimensional structure, the three families encode similar enzymatic and biological functions. Recent studies have revealed that many immunophilins possess a chaperone function independent of PPIase activity. Knockout animal studies have confirmed multiple essential roles of immunophilins in physiology and development. An immunophilin is indeed a natural ‘protein-philin’ (Greek ‘philin’ = friend) that interacts with proteins to guide their proper folding and assembly. Received: 7 May 2006; received after revision 3 July 2006; accepted 24 August 2006     

Fractal symmetry of protein interior: what have we learned?

The application of fractal dimension-based constructs to probe the protein interior dates back to the development of the concept of fractal dimension itself. Numerous approaches have been tried and tested over a course of (almost) 30 years with the aim of elucidating the various facets of symmetry of self-similarity prevalent in the protein interior. In the last 5 years especially, there has been a startling upsurge of research that innovatively stretches the limits of fractal-based studies to present an array of unexpected results on the biophysical properties of protein interior. In this article, we introduce readers to the fundamentals of fractals, reviewing the commonality (and the lack of it) between these approaches before exploring the patterns in the results that they produced. Clustering the approaches in major schools of protein self-similarity studies, we describe the evolution of fractal dimension-based methodologies. The genealogy of approaches (and results) presented here portrays a clear picture of the contemporary state of fractal-based studies in the context of the protein interior. To underline the utility of fractal dimension-based measures further, we have performed a correlation dimension analysis on all of the available non-redundant protein structures, both at the level of an individual protein and at the level of structural domains. In this investigation, we were able to separately quantify the self-similar symmetries in spatial correlation patterns amongst peptide–dipole units, charged amino acids, residues with the π-electron cloud and hydrophobic amino acids. The results revealed that electrostatic environments in the interiors of proteins belonging to ‘α/α toroid’ (all-α class) and ‘PLP-dependent transferase-like’ domains (α/β class) are highly conducive. In contrast, the interiors of ‘zinc finger design’ (‘designed proteins’) and ‘knottins’ (‘small proteins’) were identified as folds with the least conducive electrostatic environments. The fold ‘conotoxins’ (peptides) could be unambiguously identified as one type with the least stability. The same analyses revealed that peptide–dipoles in the α/β class of proteins, in general, are more correlated to each other than are the peptide–dipoles in proteins belonging to the all-α class. Highly favorable electrostatic milieu in the interiors of TIM-barrel, α/β-hydrolase structures could explain their remarkably conserved (evolutionary) stability from a new light. Finally, we point out certain inherent limitations of fractal constructs before attempting to identify the areas and problems where the implementation of fractal dimension-based constructs can be of paramount help to unearth latent information on protein structural properties.     

DNA hypomethylation in the origin and pathogenesis of human diseases

The pathogenesis of any given human disease is a complex multifactorial process characterized by many biologically significant and interdependent alterations. One of these changes, specific to a wide range of human pathologies, is DNA hypomethylation. DNA hypomethylation signifies one of the major DNA methylation states that refers to a relative decrease from the “normal” methylation level. It is clear that disease by itself can induce hypomethylation of DNA; however, a decrease in DNA methylation can also have an impact on the predisposition to pathological states and disease development. This review presents evidence suggesting the involvement of DNA hypomethylation in the pathogenesis of several major human pathologies, including cancer, atherosclerosis, Alzheimer’s disease, and psychiatric disorders. The views expressed in this paper do not necessarily represent those of the US Food and Drug Administration.     

Kallmann’s syndrome, a neuronal migration defect

Infertility and inability to smell are the phenotypical features of Kallmann’s syndrome (KS), a genetic disease which affects 1 in 10,000 males and 1 in 50,000 females, the majority of the cases being sporadic. The molecular pathogenesis of KS is complex but mainly referable to the impairment of olfactory axon development and of the migration of gonadotropin-releasing hormone (GnRH) neurons. Only two different genes have been identified so far as responsible for the disease: KAL1 and KAL2, encoding anosmin-1 and fibroblast growth factor receptor 1 (FGFR1), respectively. In this review we focus our attention on insights evoked by recent studies, which propose a new direct role for anosmin-1 in the migration GnRH neurons, and a fascinating hypothesis of interactions between anosmin-1 and FGFR1 systems. Received 23 December 2005; received after revision 31 May 2006; accepted 6 July 2006     

Mitochondrial dynamics in cell death and neurodegeneration

Mitochondria are highly dynamic organelles that continuously undergo two opposite processes, fission and fusion. Mitochondrial dynamics influence not only mitochondrial morphology, but also mitochondrial biogenesis, mitochondrial distribution within the cell, cell bioenergetics, and cell injury or death. Drp1 mediates mitochondrial fission, whereas Mfn1/2 and Opa1 control mitochondrial fusion. Neurons require large amounts of energy to carry out their highly specialized functions. Thus, mitochondrial dysfunction is a prominent feature in a variety of neurodegenerative diseases. Mutations of Mfn2 and Opa1 lead to neuropathies such as Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. Moreover, both Aβ peptide and mutant huntingtin protein induce mitochondrial fragmentation and neuronal cell death. In addition, mutants of Parkinson’s disease-related genes also show abnormal mitochondrial morphology. This review highlights our current understanding of abnormal mitochondrial dynamics relevant to neuronal synaptic loss and cell death in neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.