Myosin I: From yeast to human

Myosin I is a non-filamentous, single-headed, actin-binding motor protein and is present in a wide range of species from yeast to man. The role of these class I myosins have been studied extensively in simple eukaryotes, showing their role in diverse processes such as actin cytoskeleton organization, cell motility, and endocytosis. Recently, studies in metazoans have begun to reveal more specialized functions of myosin I. It will be a major challenge in the future to examine the physiological functions of each class I myosin in different cell types of metazoans.     

The utility F-box for protein destruction

A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.     

Neurotrophins and neuronal differentiation in the central nervous system

The central nervous system requires the proper formation of exquisitely precise circuits to function properly. These neuronal circuits are assembled during development by the formation of synaptic connections between hundreds of thousands of differentiating neurons. For these circuits to form correctly, neurons must elaborate precisely patterned axonal and dendritic arbors. Although the cellular and molecular mechanisms that guide neuronal differentiation and formation of connections remain mostly unknown, the neurotrophins have emerged recently as attractive candidates for regulating neuronal differentiation in the developing brain. The experiments reviewed here provide strong support for a bifunctional role for the neurotrophins in axonal and dendritic growth and are consistent with the exciting possibility that the neurotrophins might mediate activity-dependent synaptic plasticity.     

Molecular and cellular basis of small- and intermediate-conductance, calcium-activated potassium channel function in the brain

Small conductance calcium-activated potassium (SK or K_Ca2) channels link intracellular calcium transients to membrane potential changes. SK channel subtypes present different pharmacology and distribution in the nervous system. The selective blocker apamin, SK enhancers and mice lacking specific SK channel subunits have revealed multifaceted functions of these channels in neurons, glia and cerebral blood vessels. SK channels regulate neuronal firing by contributing to the afterhyperpolarization following action potentials and mediating I_AHP, and partake in a calcium-mediated feedback loop with NMDA receptors, controlling the threshold for induction of hippocampal long-term potentiation. The function of distinct SK channel subtypes in different neurons often results from their specific coupling to different calcium sources. The prominent role of SK channels in the modulation of excitability and synaptic function of limbic, dopaminergic and cerebellar neurons hints at their possible involvement in neuronal dysfunction, either as part of the causal mechanism or as potential therapeutic targets. Received 23 April 2008; received after revision 29 May 2008; accepted 4 June 2008     

In situ demonstration of the activity of 3β-hydroxysteroid dehydrogenase on steroid hormone secreting cells within the paraaortic lymph nodes of golden hamster

Summary The in situ activity of 3-hydroxysteroid dehydrogenase was detected in clustered cells which showed steroid-producing morphology, within the capsule of the paraaortic lymph node. In light and electron microscopic studies, the positive reaction products were detected on intracapsular cell clusters. This result indicates that these unique cells may have a steroid secreting function within the lymph nodes.     

KIF5C: A new binding partner for protein kinase CK2 with a preference for the CK2α’ subunit

Protein kinase CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in eukaryotic cells. CK2 is a constitutively active tetrameric enzyme composed of two catalytic α and/or α’-subunits and two regulatory β-subunits. There is increasing evidence that the individual subunits may have independent functions and that they are asymmetrically distributed inside the cell. To gain a better understanding of the functions of the individual subunits, we employed a yeast-two-hybrid screen with CK2α and CK2α’. We identified the motor neuron protein KIF5C as a new binding partner for CK2. The interaction found in the yeast-two-hybrid screen was confirmed by co-sedimentation analysis on a sucrose density gradient and by co-immunoprecipitation analysis. Pull-down experiments and surface plasmon resonance spectrometry revealed a direct binding of KIF5C to CK2α’. Co-localization studies with neuroblastoma cells, bone marrow and with primary neurons confirmed the biochemical analysis that KIF5C preferentially bound to CK2α’. Received 8 August 2008; received after revision 3 November 2008; accepted 4 November 2008     

Cbln and C1q family proteins – New transneuronal cytokines

The C1q family is characterized by a C-terminal conserved global C1q domain, which is structurally very similar to the tumor necrosis factor homology domain. Although some C1q family members are expressed in the central nervous system, their functions have not been well characterized. Cbln1, a member of the Cbln subfamily of the C1q family, is predominantly expressed in cerebellar granule cells. Interestingly, Cbln1 was recently shown to play two unique roles at excitatory synapses formed between cerebellar granule cells and Purkinje cells: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytosis pathway. Since other Cbln subfamily members, Cbln2-Cbln4, are expressed in various regions of developing and mature brains, Cbln subfamily proteins may generally serve as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions.     

The structure and functions of the presenilins

The presenilins (PSs) were new proteins discovered in 1995 to be involved, among other functions, in the molecular mechanisms leading to Alzheimers disease. These proteins have been the subject of many investigations since then to elucidate their molecular structures and functions. Until now, the conclusions about PS structure have been discordant, but the 8-TM structure has been accepted by the Alzheimers community, with the evidence for the 7-TM structure largely ignored. Here the evidence is reviewed for the 6-TM, 7-TM, 8-TM and other proposed models of PS topography and possibilities offered for the differences in interpretation of the various sets of data. The conclusion is that at this stage, the 7-TM model for cell surface PS is most likely the correct one.Received 22 December 2004; accepted 26 January 2005     

Neuroreplacement therapy and stem cell biology under disease conditions

Recent advances in stem cell technology are expanding our ability to replace a variety of cells throughout the body. In the past, neurological diseases caused by the degeneration of neuronal cells were considered incurable because of a long-held 'truism'; neurons do not regenerate during adulthood. However, this statement has been challenged, and we have now found much evidence that the brain is indeed capable of regenerating neurons after maturing. Based on this new concept, researchers have shown neural differentiation of stem cells and recovery of function following transplantation of these cells into the brain. These results may promise a bright future for clinical applications of stem cell strategies in neurological diseases; however, we must consider the pathophysiological environments of individual diseases that may affect stem cell biology. Before we begin to develop clinical applications, we must consider environmental factors that have not been discussed in the current preclinical studies. Here, we study cases of Alzheimer's disease and schizophrenia and discuss the effects of environmental factors under disease conditions.Received 15 January 2003; received after revision 7 April 2003; accepted 8 April 2003     

Proliferating cell nuclear antigen: a proteomics view

Proliferating cell nuclear antigen (PCNA), a cell cycle marker protein, is well known as a DNA sliding clamp for DNA polymerase delta and as an essential component for eukaryotic chromosomal DNA replication and repair. Due to its mobility inside nuclei, PCNA is dynamically presented in a soluble or chromatin-associated form. The heterogeneity and specific modifications of PCNA may reflect its multiple functions and the presence of many binding partners in the cell. The recent proteomics approaches applied to characterizing PCNA interactions revealed multiple PCNA partners with a wide spectrum of activity and unveiled the possible existence of new PCNA functions. Since more than 100 PCNA-interacting proteins and several PCNA modifications have already been reported, a proteomics point of view seems exactly suitable to better understand the role of PCNA in cellular functions. Received 29 May 2008; received after revision 7 July 2008; accepted 16 July 2008     

Muscle LIM protein promotes expression of the acetylcholine receptor <Emphasis Type="Italic">γ</Emphasis>-subunit gene cooperatively with the myogenin-E12 complex

Muscle LIM protein (MLP, also referred to as CRP3) is a muscle-specific LIM-only protein, which consists of two LIM motifs. MLP functions as a positive regulator during myogenesis. Here we report that MLP serves as a cofactor regulating the expression of the nicotinic acetylcholine receptor (AChR) -subunit gene in skeletal muscle cells. We found that MLP promoted the expression of the AChR -subunit gene in C2C12 myotubes, but not in C2C12 myoblasts or NIH3T3 fibroblasts. Furthermore, we showed that MLP interacted with myogenin in vivo and enhanced the binding ability of the myogenin-E12 heterodimer to the E boxes in the AChR -subunit gene promoter. Together, these results suggest that MLP promotes the specific expression of the AChR -subunit gene cooperatively with the myogenin-E12 complex during myogenesis.Received 17 May 2004; received after revision 22 July 2004; accepted 26 July 2004     

Collapsin response mediator protein-2 is a calmodulin-binding protein

Collapsin response mediator protein-2 (CRMP-2) plays a crucial role in axonal guidance and neurite outgrowth during neural development and regeneration. We have studied the interaction between calmodulin (CaM) and CRMP-2 and how Ca²⁺/CaM binding modulates the biological functions of CRMP-2. We have shown that CRMP-2 binds to CaM directly in a Ca²⁺-dependent manner. The CaM binding site of CRMP-2 is proposed to reside in the last helix of the folded domain, and in line with this, a synthesized peptide representing this helix bound to CaM. In addition, CaM binding inhibits a homotetrameric assembly of CRMP-2 and attenuates calpainmediated CRMP-2 proteolysis. Furthermore, a CaM antagonist reduces the number and length of process induced by CRMP-2 overexpression in HEK293 cells. Take together, our data suggest that CRMP-2 is a novel CaM-binding protein and that CaM binding may play an important role in regulating CRMP-2 functions. Received 26 June 2008; received after revision 18 November 2008; accepted 24 November 2008     

Histone methylation and ubiquitination with their cross-talk and roles in gene expression and stability

Methylation of lysine residues of histones is associated with functionally distinct regions of chromatin, and, therefore, is an important epigenetic mark. Over the past few years, several enzymes that catalyze this covalent modification on different lysine residues of histones have been discovered. Intriguingly, histone lysine methylation has also been shown to be cross-regulated by histone ubiquitination or the enzymes that catalyze this modification. These covalent modifications and their cross-talks play important roles in regulation of gene expression, heterochromatin formation, genome stability, and cancer. Thus, there has been a very rapid progress within past several years towards elucidating the molecular basis of histone lysine methylation and ubiquitination, and their aberrations in human diseases. Here, we discuss these covalent modifications with their cross-regulation and roles in controlling gene expression and stability. Received 24 September 2008; received after revision 21 November 2008; accepted 28 November 2008     

Structural insights into the multiple functions of protein C inhibitor

Protein C inhibitor (PCI) is a widely distributed, multifunctional member of the serpin family of protease inhibitors, and has been implicated in several physiological processes and disease states. Its inhibitory activity and specificity are regulated by binding to cofactors such as heparin, thrombomodulin and phospholipids, and it also appears to have non-inhibitory functions related to hormone and lipid binding. Just how the highly conserved serpin architecture can support the multiple diverse functions of PCI is a riddle best addressed by protein crystallography. Over the last few years we have solved the structure of PCI in its native, cleaved and protein-complexed states. They reveal a conserved serpin fold and general mechanism of protease inhibition, but with some unique features relating to inhibitory specificity/promiscuity, cofactor binding and hydrophobic ligand transport. Received 1 July 2008; received after revision 16 August 2008; accepted 22 August 2008     

Bovine microvascular endothelial cells of separate morphology differ in growth and response to the action of interferon-γ

Five cell types recently isolated from the bovine corpus luteum differed in their epithelioid morphology and their cytoskeleton, but shared common criteria of microvascular endothelial cells^1,2. To give strong evidence for the separate entity, the growth rate of the 5 phenotypically different cells was studied. They were seeded at low density on day 0. Most of these cells were treated with 200 to 1000 U recombinant bovine interferon- (IFN-) for 3 days. The untreated remainder served as controls. Cell counts were made for all cultures on days 4, 7, 10 and 13. morphology: 13 d after treatment with IFN- senescent cells as well as intact cells occurred in cultures of cell types 1 to 4. Cultures of cell type 5 were apparently unchanged and resembled their untreated counterparts. Desminpositive cells in cultures of cell type 2 developed cell processes. Growth rate: In the absence of IFN-, the growth rate was high for cell types 3 and 4, moderate for cell type 1, and low for cell types 2 and 5. The presence of IFN- caused anti-proliferative effects. These were higher for cell types 3 and 4 than for cell types 1 and 2. IFN- could be cytotoxic on cell type 3. In contrast, the cytokine tended to support the cell growth of cell type 5. These findings substantiate the postulate that endothelial cells exhibiting separate morphology in culture also function differently.     

Role of full-length osteoprotegerin in tumor cell biology

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22–194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted. Received 2 September 2008; received after revision 29 September 2008; accepted 13 October 2008