RESUMEN

Human papillomavirus (HPV) is associated with infection of different tissues, such as the cervix, anus, vagina, penis, vulva, oropharynx, throat, tonsils, back of the tongue, skin, the lungs, among other tissues. HPV infection may or may not be associated with the development of cancer, where HPVs not related to cancer are defined as low-risk HPVs and are associated with papillomatosis disease. In contrast, high-risk HPVs (HR-HPVs) are associated with developing cancers in areas that HR-HPV infects, such as the cervix. In general, infection of HPV target cells is regulated by specific molecules and receptors that induce various conformational changes of HPV capsid proteins, allowing activation of HPV endocytosis mechanisms and the arrival of the HPV genome to the human cell nucleus. After the transcription of the HPV genome, the HPV genome duplicates exponentially to lodge in a new HPV capsid, inducing the process of exocytosis of HPV virions and thus releasing a new HPV viral particle with a high potential of infection. This infection process allows the HPV viral life cycle to conclude and enables the growth of HPV virions. Understanding the entire infection process has been a topic that researchers have studied and developed for decades; however, there are many things to still understand about HPV infection. A thorough understanding of these HPV infection processes will allow new potential treatments for HPV-associated cancer and papillomatosis. This chapter focuses on HPV infection, the process that will enable HPV to complete its HPV life cycle, emphasizing the critical role of different molecules in allowing this infection and its completion during the HPV viral life cycle.

Asunto(s)

Papiloma , Infecciones por Papillomavirus , Masculino , Femenino , Humanos , Infecciones por Papillomavirus/complicaciones , Virus del Papiloma Humano , Proteínas de la Cápside/genética , Papiloma/complicaciones , Piel

RESUMEN

Introduction: Coccidiosis, caused by parasites of numerous Eimeria species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine. Methods: This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of E. tenella were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite E. tenella serum assessed. The selected peptide clones inferred amino acid sequences matched numerous E. tenella proteins. Results and Conclusions: The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of E. tenella micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of E. tenella was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative E. tenella protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected E. tenella native antigens. Discussion: Thus, selected phagotopes contained recombinant E. tenella antigen peptides. Using antibodies against E. tenella sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an E. tenella recombinant epitope-based vaccine.

RESUMEN

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

RESUMEN

Zika virus (ZIKV) infection is a major public health threat, making the study of its biology a matter of great importance. By analyzing the viral-host protein interactions, new drug targets may be proposed. In this work, we showed that human cytoplasmic dynein-1 (Dyn) interacts with the envelope protein (E) of ZIKV. Biochemical evidence indicates that the E protein and the dimerization domain of the heavy chain of Dyn binds directly without dynactin or any cargo adaptor. Analysis of this interactions in infected Vero cells by proximity ligation assay suggest that the E-Dyn interaction is dynamic and finely tuned along the replication cycle. Altogether, our results suggest new steps in the replication cycle of the ZIKV for virion transport and indicate a suitable molecular target to modulate infection by ZIKV.

Asunto(s)

Infección por el Virus Zika , Virus Zika , Chlorocebus aethiops , Humanos , Animales , Dineínas Citoplasmáticas , Células Vero , Transporte Biológico

RESUMEN

Glucocorticoid steroids play cardinal roles during the life span of an individual, modulating almost all aspects of the physiology, including the metabolism of carbohydrates, lipids and amino acids, as well as the immune response, neurological biology, stress adaptation, apoptosis, cell division, cell fate, inflammatory responses, etc. Glucocorticoids exert their biological effects by activation of the glucocorticoid receptor (GR), a bona fide ligand-activated transcription factor belonging to the nuclear receptor superfamily. The GR is expressed in virtually all cells of the human body showing isoformic versions and also transcription variants. GR forms oligomeric heterocomplexes that include the 90-kDa heat-shock protein (Hsp90) as an essential hub of the chaperone oligomer. The nature of chaperones associated with this heterocomplex is responsible for the modulation of the subcellular localization of the GR and its biological actions in a given tissue or cell type. In this sense, the discovery that immunophilins containing tetratricopeptide repeats (TPR) domains are responsible for the GR cytoplasmic transport mechanism and the nuclear retention half-time of the receptor opened new trends in our understanding of its complex mechanism of action. Because the properties of GR ligands influence these protein-protein interactions, specific steroid•receptor complexes may confer the GR different features providing new therapeutic opportunities to manage the disease. In this article, we analyze multiple aspects of the GR mechanism of action, some properties of the GR isoforms, and the latest findings revealing the roles of Hsp90-binding immunophilins to manage the glucocorticoid biological response.

Asunto(s)

Glucocorticoides , Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/química , Glucocorticoides/farmacología , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Isoformas de Proteínas

RESUMEN

Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.

Asunto(s)

Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Receptor trkB , Ratones , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Receptor trkB/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cuerpo Celular , Neuronas/fisiología , Axones/metabolismo , Endosomas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Transposition of the great arteries (TGA) is a congenital heart defect with a complex pathogenesis that has not been fully elucidated. In this study, we performed whole-exome sequencing (WES) in isolated TGA-diagnosed patients and analyzed genes of motile and non-motile cilia ciliogenesis and ciliary trafficking, as well as genes previously associated with this heart malformation. Deleterious missense and splicing variants of genes DNAH9, DNAH11, and ODAD4 of cilia outer dynein arm and central apparatus, HYDIN, were found in our TGA patients. Remarkable, there is a clustering of deleterious genetic variants in cilia genes, suggesting it could be an oligogenic disease. Our data evidence the genetic diversity and etiological complexity of TGA and point out that population allele determination and genetic aggregation studies are required to improve genetic counseling.

Asunto(s)

Cilios , Transposición de los Grandes Vasos , Arterias , Dineínas Axonemales/genética , Cilios/genética , Análisis por Conglomerados , Humanos , Transposición de los Grandes Vasos/genética , Secuenciación del Exoma

RESUMEN

Neurons are highly polarized cells that rely on the intracellular transport of organelles. This process is regulated by molecular motors such as dynein and kinesins and the Rab family of monomeric GTPases that together help move cargo along microtubules in dendrites, somas, and axons. Rab5-Rab11 GTPases regulate receptor trafficking along early-recycling endosomes, which is a process that determines the intracellular signaling output of different signaling pathways, including those triggered by BDNF binding to its tyrosine kinase receptor TrkB. BDNF is a well-recognized neurotrophic factor that regulates experience-dependent plasticity in different circuits in the brain. The internalization of the BDNF/TrkB complex results in signaling endosomes that allow local signaling in dendrites and presynaptic terminals, nuclear signaling in somas and dynein-mediated long-distance signaling from axons to cell bodies. In this review, we briefly discuss the organization of the endocytic pathway and how Rab11-recycling endosomes interact with other endomembrane systems. We further expand upon the roles of the Rab11-recycling pathway in neuronal plasticity. Then, we discuss the BDNF/TrkB signaling pathways and their functional relationships with the postendocytic trafficking of BDNF, including axonal transport, emphasizing the role of BDNF signaling endosomes, particularly Rab5-Rab11 endosomes, in neuronal plasticity. Finally, we discuss the evidence indicating that the dysfunction of the early-recycling pathway impairs BDNF signaling, contributing to several neurodegenerative diseases.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo , Enfermedades Neurodegenerativas , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Dineínas/metabolismo , Endosomas/metabolismo , GTP Fosfohidrolasas/metabolismo , Hipocampo/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Transporte de Proteínas , Receptor trkB , Proteínas de Unión al GTP rab

RESUMEN

Steroid receptors form soluble heterocomplexes with the 90-kDa heat-shock protein (Hsp90) and other chaperones and co-chaperones. The assembly and composition of the oligomer is influenced by the presence and nature of the bound steroid. Although these receptors shuttle dynamically in and out of the nucleus, their primary localization in the absence of steroid can be mainly cytoplasmic, mainly nuclear, or partitioned into both cellular compartments. Upon steroid binding, receptors become localized to the nucleus via the transportosome, a retrotransport molecular machinery that comprises Hsp90, a high-molecular-weight immunophilin, and dynein motors. This molecular machinery, first evidenced in steroid receptors, can also be used by other soluble proteins. In this review, we dissect the complete model of this transport machinery system.

Asunto(s)

Inmunofilinas , Receptores de Esteroides , Núcleo Celular , Proteínas HSP90 de Choque Térmico , Humanos , Chaperonas Moleculares , Receptores de Glucocorticoides

Asunto(s)

Trastornos de la Motilidad Ciliar , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/terapia , Humanos

RESUMEN

Following entry into the host cell, retroviruses generate a dsDNA copy of their genomes via reverse transcription, and this viral DNA is subsequently integrated into the chromosomal DNA of the host cell. Before integration can occur, however, retroviral DNA must be transported to the nucleus as part of a 'preintegration complex' (PIC). Transporting the PIC through the crowded environment of the cytoplasm is challenging, and retroviruses have evolved different mechanisms to accomplish this feat. Within a eukaryotic cell, microtubules act as the roads, while the microtubule-associated proteins dynein and kinesin are the vehicles that viruses exploit to achieve retrograde and anterograde trafficking. This review will examine the various mechanisms retroviruses have evolved in order to achieve retrograde trafficking, confirming that each retrovirus has its own strategy to functionally subvert microtubule associated proteins.

Asunto(s)

Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Retroviridae/fisiología , Transporte Biológico , Dineínas/química , Dineínas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Replicación Viral

RESUMEN

Lysosomes are dynamic organelles, which can fuse with a variety of targets and undergo constant regeneration. They can move along microtubules in a retrograde and anterograde fashion by using motor proteins, kinesin and dynein, being main players in extracellular secretion, intracellular components degradation and recycling. Moreover, lysosomes interact with other intracellular organelles to regulate their turnover, such as ER, mitochondria and peroxisomes. The correct localization of lysosomes is relevant in several physiological processes, including appropriate antigen presentation, neurotransmission and receptors modulation in neuronal synapsis, whereas hepatic lysosomes and autophagy are master regulators of nutrient homeostasis. Alterations in lysosome function due to mutation of genes encoding lysosomal proteins, soluble hydrolases as well as membrane proteins, lead to lysosomal storage diseases (LSDs). Lysosomes containing undegraded substrates are finally stacked and therefore miss positioned inside the cell, leading to lysosomal dysfunction, which impacts a wide range of cellular functions.

Asunto(s)

Movimiento Celular , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Redes y Vías Metabólicas/genética , Modelos Biológicos , Mutación , Proteínas/genética

RESUMEN

Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects.

Asunto(s)

Proteínas HSP90 de Choque Térmico/metabolismo , Inmunofilinas/metabolismo , Animales , Proteínas HSP90 de Choque Térmico/química , Humanos , Inmunofilinas/química , Modelos Moleculares , Estructura Molecular , Unión Proteica

RESUMEN

The success of viruses in the delivery of the viral genome to target cells relies on the evolutionary selection of protein-based domains able to hijack the intermolecular interactions through which cells respond to intra- and extracellular stimuli. In an effort to mimic viral infection capabilities during non-viral gene delivery, a modular recombinant protein named T-Rp3 was recently developed, containing a DNA binding domain, a dynein molecular motor interacting domain, and a TAT-derived transduction domain. Here, we analyzed at the microscopic level the mechanisms behind the cell internalization and intracellular trafficking of this highly efficient modular protein vector. We found that the protein has the ability to self-assemble in discrete protein nanoparticles resembling viral capsids, to bind and condense plasmid DNA (pDNA), and to interact with eukaryotic cell membranes. Confocal and single particle tracking assays performed on living HeLa cells revealed that the T-Rp3 nanoparticles promoted an impressive speed of cellular uptake and perinuclear accumulation. Finally, the protein demonstrated to be a versatile vector, delivering siRNA at efficiencies comparable to Lipofectamine™. These results demonstrate the high potential of recombinant modular proteins with merging biological functions to fulfill several requirements needed to obtain cost-effective non-viral vectors for gene-based therapies.

Asunto(s)

Dineínas/administración & dosificación , Técnicas de Transferencia de Gen , Nanopartículas/administración & dosificación , ADN/administración & dosificación , Escherichia coli/genética , Células HeLa , Humanos , Plásmidos , Dominios Proteicos/genética , ARN Interferente Pequeño/administración & dosificación , Proteínas Recombinantes/genética

RESUMEN

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor dysfunction, cognitive deficits, and psychiatric symptoms. The primary genetic cause is an expansion of cytosine adenine guanine (CAG) nucleotides of the huntingtin gene, which codes an important protein involved with neuronal signaling. The severity of HD correlates with the number of CAG repeats and individuals with longer expansions have an earlier onset and more severe symptoms. A microarray study conducted by our research group showed alteration in DNAH6 gene (encoding dynein axonemal heavy chain 6). DNAH6 belongs to dynein family, whose members are constituents of the microtubule-associated motor proteins and is downregulated in the striatum of a HD mouse model (knockin HdhQ111/Q111). In this manner, our goal was to confirm these downregulations in the mouse model and verify if the same alteration in the axonemal DNAH6 gene expression is observed in blood samples of HD patients. Blood samples were collected from 17 patients with clinical diagnosis of HD and 12 healthy individuals and RNA extracted for qPCR analysis. Microarray data were confirmed by qPCR in knockin HdhQ111/Q111, and DNAH6 was severely decreased in those mice, as compared to control mice (HdhQ20/Q20). Notably, decreased expression of DNAH6 gene was also observed in HD patients when compared to control group and negatively correlates with the CAG expansion. Although further studies are necessary to underlie the molecular mechanisms of dynein-htt interaction, this data highlights DNAH6 as a potential new blood marker for HD.

Asunto(s)

Dineínas/sangre , Enfermedad de Huntington/sangre , Animales , Biomarcadores/sangre , Estudios de Casos y Controles , Regulación hacia Abajo , Dineínas/genética , Dineínas/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Investigación Biomédica Traslacional

RESUMEN

How murine leukemia virus (MLV) travels from the cell membrane to the nucleus and the mechanism for nuclear entry of MLV DNA in dividing cells still remain unclear. It seems likely that the MLV preintegration complex (PIC) interacts with cellular proteins to perform these tasks. We recently published that the microtubule motor cytoplasmic dynein complex and its regulator proteins interact with the MLV PIC at early times of infection, suggesting a functional interaction between the incoming viral particles, the dynein complex, and dynein regulators. To better understand the role of the dynein complex in MLV infection, we performed short hairpin RNA (shRNA) screening of the dynein light chains on MLV infection. We found that silencing of a specific light chain of the cytoplasmic dynein complex, DYNLRB2, reduced the efficiency of infection by MLV reporter viruses without affecting HIV-1 infection. Furthermore, the overexpression of DYNLRB2 increased infection by MLV. We conclude that the DYNLRB2 light chain of the cytoplasmic dynein complex is an important and specific piece of the host machinery needed for MLV infection.IMPORTANCE Retroviruses must reach the chromatin of their host to integrate their viral DNA, but first they must get into the nucleus. The cytoplasm is a crowded environment in which simple diffusion is slow, and thus viruses utilize retrograde transport along the microtubule network, mediated by the dynein complex. Different viruses use different components of this multisubunit complex. We have found that murine leukemia virus (MLV) associates functionally and specifically with the dynein light chain DYNLRB2, which is required for infection. Our study provides more insight into the molecular requirements for retrograde transport of the MLV preintegration complex and demonstrates, for the first time, a role for DYNLRB2 in viral infection.

Asunto(s)

Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/fisiología , Interacciones Huésped-Patógeno , Virus de la Leucemia Murina/fisiología , Animales , Transporte Biológico , Línea Celular , Núcleo Celular/virología , Células HEK293 , VIH-1/fisiología , Interacciones Huésped-Patógeno/genética , Humanos , Ratones , Microtúbulos/virología , Células 3T3 NIH

RESUMEN

The Kunitz-type recombinant protein, Amblyomin-X, is an antitumor recombinant molecule from a cDNA library prepared from the salivary glands of the tick Amblyomma cajennense. The primary target of this protein appears to be the proteasome. Amblyomin-X increased gene and protein expression of distinct subunits of the molecular motor dynein, which plays a key role in the intracellular transport. Herein, Amblyomin-X was specifically taken up by tumor cells through lipid-raft endocytic pathways, but not by fibroblasts. Moreover, dynein inhibitor, ciliobrevin A, decreased Amblyomin-X uptake by tumor cells. Furthermore, incubation of tumor cells with Amblyomin-X inhibited trypsin-like activity of the proteasome, which was restored upon pretreatment with ciliobrevin A. Only in tumor cells treated with Amblyomin-X, we identified proteins bounds to dynein that are related to aggresome formation, autophagy inhibition, and early and recycling endosome markers. In addition, Amblyomin-X was found to interact with dynein, increased Rab11A protein expression and Rab11A co-localization with the light-intermediate chain 2 (LIC2) of dynein. Thereby, the results provide new insights on the antitumor mechanism of Amblyomin-X and reveal an unsuspected role of cytoplasmic dynein in its uptake, intracellular trafficking and pro-apoptotic action.

Asunto(s)

Apoptosis/efectos de los fármacos , Dineínas Citoplasmáticas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas y Péptidos Salivales/farmacología , Animales , Apoptosis/fisiología , Proteínas de Artrópodos , Autofagia/fisiología , Línea Celular Tumoral , Humanos , Proteínas Recombinantes/metabolismo , Garrapatas

RESUMEN

PPARγ is a ligand-activated nuclear receptor best known for its involvement in adipogenesis and glucose homeostasis. PPARγ activity has also been associated with neuroprotection in different neurological disorders, but the mechanisms involved in PPARγ effects in the nervous system are still unknown. Here we describe a new functional role for PPARγ in neuronal responses to injury. We found both PPAR transcripts and protein within sensory axons and observed an increase in PPARγ protein levels after sciatic nerve crush. This was correlated with increased retrograde transport of PPARγ after injury, increased association of PPARγ with the molecular motor dynein, and increased nuclear accumulation of PPARγ in cell bodies of sensory neurons. Furthermore, PPARγ antagonists attenuated the response of sensory neurons to sciatic nerve injury, and inhibited axonal growth of both sensory and cortical neurons in culture. Thus, axonal PPARγ is involved in neuronal injury responses required for axonal regeneration. Since PPARγ is a major molecular target of the thiazolidinedione (TZD) class of drugs used in the treatment of type II diabetes, several pharmaceutical agents with acceptable safety profiles in humans are available. Our findings provide motivation and rationale for the evaluation of such agents for efficacy in central and peripheral nerve injuries.

Asunto(s)

Axones/metabolismo , Regulación de la Expresión Génica/fisiología , Regeneración Nerviosa/fisiología , Neuronas/patología , PPAR gamma/metabolismo , Neuropatía Ciática/patología , Anilidas/farmacología , Animales , Axones/efectos de los fármacos , Axotomía , Células Cultivadas , Embrión de Mamíferos , Ganglios Espinales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neurofilamentos/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley

RESUMEN

Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+).

Asunto(s)

Dineínas Axonemales/metabolismo , Cadmio/toxicidad , ATPasas Transportadoras de Calcio/metabolismo , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Cadmio/administración & dosificación , Cadmio/farmacocinética , Membrana Celular/enzimología , Relación Dosis-Respuesta a Droga , Pruebas de Enzimas , Voluntarios Sanos , Humanos , Masculino , Espermatozoides/enzimología

RESUMEN

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1.

Asunto(s)

1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Proteínas Fúngicas/genética , Proteínas Asociadas a Microtúbulos/genética , Neurospora crassa/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa/química , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Complejo Dinactina , Dineínas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expresión Génica , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Neurospora crassa/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Alineación de Secuencia