Role of J-domain Proteins in Yeast Physiology and Protein Quality Control.

The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the folding of newly synthesized proteins to the disassembly and refolding of protein aggregates. This multifunctionality of Hsp70 is governed by J-domain proteins (JDPs), which act as indispensable co-chaperones that target specific substrates to Hsp70. The number of distinct JDPs present in a species always outnumbers Hsp70, documenting JDP function in functional diversification of Hsp70. In this review, we describe the physiological roles of JDPs in the Saccharomyces cerevisiae PQC system, with a focus on the abundant JDP generalists, Zuo1, Ydj1 and Sis1, which function in fundamental cellular processes. Ribosome-bound Zuo1 cooperates with the Hsp70 chaperones Ssb1/2 in folding and assembly of nascent polypeptides. Ydj1 and Sis1 cooperate with the Hsp70 members Ssa1 to Ssa4 to exert overlapping functions in protein folding and targeting of newly synthesized proteins to organelles including mitochondria and facilitating the degradation of aberrant proteins by E3 ligases. Furthermore, they act in protein disaggregation reactions, though Ydj1 and Sis1 differ in their modes of Hsp70 cooperation and substrate specificities. This results in functional specialization as seen in prion propagation and the underlying dominant role of Sis1 in targeting Hsp70 for shearing of prion amyloid fibrils.

The cytoprotective sequestration activity of small heat shock proteins is evolutionarily conserved.

The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants. We show that sequestration of misfolded proteins is an original and widespread activity among sHsps executed by specific family members. Sequestrase positive C. elegans' sHsps harbor specific sequence features, including a high content of aromatic and methionine residues in disordered N-terminal extensions. Those sHsps buffer limitations in Hsp70 capacity in C. elegans WT animals and are upregulated in long-lived daf-2 mutants, contributing to lifespan extension. Cellular protection by sequestration of misfolded proteins is, therefore, an evolutionarily conserved activity of the sHsp family.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Chaperone-Mediated Protein Disaggregation Triggers Proteolytic Clearance of Intra-nuclear Protein Inclusions.

The formation of insoluble inclusions in the cytosol and nucleus is associated with impaired protein homeostasis and is a hallmark of several neurodegenerative diseases. Due to the absence of the autophagic machinery, nuclear protein aggregates require a solubilization step preceding degradation by the 26S proteasome. Using yeast, we identify a nuclear protein quality control pathway required for the clearance of protein aggregates. The nuclear J-domain protein Apj1 supports protein disaggregation together with Hsp70 but independent of the canonical disaggregase Hsp104. Disaggregation mediated by Apj1/Hsp70 promotes turnover rather than refolding. A loss of Apj1 activity uncouples disaggregation from proteasomal turnover, resulting in accumulation of toxic soluble protein species. Endogenous substrates of the Apj1/Hsp70 pathway include both nuclear and cytoplasmic proteins, which aggregate inside the nucleus upon proteotoxic stress. These findings demonstrate the coordinated activity of the Apj1/Hsp70 disaggregation system with the 26S proteasome in facilitating the clearance of toxic inclusions inside the nucleus.

Conidiation in Neurospora crassa: vegetative reproduction by a model fungus.

Asexual development, conidiation, in the filamentous fungus Neurospora crassa is a simple developmental process that starts with the growth of aerial hyphae. Then, the formation of constrictions and subsequent maturation gives rise to the mature conidia that are easily dispersed by air currents. Conidiation is regulated by environmental factors such as light, aeration and nutrient limitation, and by the circadian clock. Different regulatory proteins acting at different stages of conidiation have been described. The role of transcription factors such as FL, and components of signal transduction pathways such as the cAMP phosphodiesterase ACON-2 suggest a complex interplay between differential transcription and signal transduction pathways. Comparisons between the molecular basis of conidiation in N. crassa and other filamentous fungi will help to identify common regulatory elements.

Cellular sequestrases maintain basal Hsp70 capacity ensuring balanced proteostasis.

Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.

Cellular Functions and Mechanisms of Action of Small Heat Shock Proteins.

Small heat shock proteins (sHsps) constitute a diverse chaperone family that shares the α-crystallin domain, which is flanked by variable, disordered N- and C-terminal extensions. sHsps act as the first line of cellular defense against protein unfolding stress. They form dynamic, large oligomers that represent inactive storage forms. Stress conditions cause a rapid increase in cellular sHsp levels and trigger conformational rearrangements, resulting in exposure of substrate-binding sites and sHsp activation. sHsps bind to early-unfolding intermediates of misfolding proteins in an ATP-independent manner and sequester them in sHsp/substrate complexes. Sequestration protects substrates from further uncontrolled aggregation and facilitates their refolding by ATP-dependent Hsp70-Hsp100 disaggregases. Some sHsps with particularly strong sequestrase activity, such as yeast Hsp42, are critical factors for forming large, microscopically visible deposition sites of misfolded proteins in vivo. These sites are organizing centers for triaging substrates to distinct quality control pathways, preferentially Hsp70-dependent refolding and selective autophagy.

Prevalence of caries and gingivitis in 2- to 4-year-old children attending daycare centers managed by the chilean national kindergartens board (JUNJI) and municipal schools, Valdivia

Objective: to determine the prevalence of carious lesions and gingivitis in 2- to 4-year-old children attending JUNJI daycare centers and urban municipal schools in the city of Valdivia, Chile. material and method: descriptive cross-sectional study. a population of 182 two-year-old children and 285 four-year-old children were examined. subjects were selected by stratified random sampling. all subjects were enrolled in daycare centers managed by JUNJI and municipal schools in the city of Valdivia. an oral examination was performed to measure the DMFT and hemorrhagic indexes according to the WHO diagnostic criteria. the presence of cavitated carious lesions and gingivitis was determined, resulting in descriptive statistics according to age and gender. results: the prevalence of caries was 12.6 percent in two-year-old children and 41 percent in four-year-olds, respectively. the prevalence of gingivitis was 36.8 percent at 2 years of age and 70.5 percent at 4 years. there were no significant differences by gender at 2 years of age (p=1) or at 4 years (p=0.37). two year-old children have significantly fewer carious lesions and gingivitis less frequently than four-year-olds (p=<.001). conclusion: two year-old children have a lower prevalence of carious lesions and gingivitis than four-year-old ones. no relationship between the variables and gender was found.

Alteration of light-dependent gene regulation by the absence of the RCO-1/RCM-1 repressor complex in the fungus Neurospora crassa.

The activation of transcription by light in the fungus Neurospora crassa requires the White Collar Complex (WCC), a photoreceptor and transcription factor complex. After light reception two WCCs interact and bind the promoters of light-regulated genes to activate transcription. This process is regulated by VVD, a small photoreceptor that disrupts the interaction between WCCs and leads to a reduction in transcription after long exposures to light. The N. crassa RCO-1/RCM-1 repressor complex is the homolog of the Tup1-Ssn6 repressor complex in yeast, and its absence modifies photoadaptation. We show that the absence of the RCO-1/RCM-1 repressor complex leads to several alterations in transcription that are gene-specific: an increase in the accumulation of mRNAs in the dark, a repression of transcription, and a derepression of transcription after long exposures to light. The absence of the RCO-1/RCM-1 repressor complex leads to lower VVD levels that are available for the regulation of the activity of the WCC. The reduction in the amount of VVD results in increased WCC binding to the promoters of light-regulated genes in the dark and after long exposures to light, leading to the modification of photoadaptation that has been observed in rco-1 and rcm-1 mutants. Our results show that the photoadaptation phenotype of mutants in the RCO-1/RCM-1 repressor complex is, at least in part, an indirect consequence of the reduction of vvd transcription, and the resulting modification in the regulation of transcription by the WCC.

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain.

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.

Regulation of conidiation by light in Aspergillus nidulans.

Light regulates several aspects of the biology of many organisms, including the balance between asexual and sexual development in some fungi. To understand how light regulates fungal development at the molecular level we have used Aspergillus nidulans as a model. We have performed a genome-wide expression analysis that has allowed us to identify >400 genes upregulated and >100 genes downregulated by light in developmentally competent mycelium. Among the upregulated genes were genes required for the regulation of asexual development, one of the major biological responses to light in A. nidulans, which is a pathway controlled by the master regulatory gene brlA. The expression of brlA, like conidiation, is induced by light. A detailed analysis of brlA light regulation revealed increased expression after short exposures with a maximum after 60 min of light followed by photoadaptation with longer light exposures. In addition to brlA, genes flbA-C and fluG are also light regulated, and flbA-C are required for the correct light-dependent regulation of the upstream regulator fluG. We have found that light induction of brlA required the photoreceptor complex composed of a phytochrome FphA, and the white-collar homologs LreA and LreB, and the fluffy genes flbA-C. We propose that the activation of regulatory genes by light is the key event in the activation of asexual development by light in A. nidulans.

A role in the regulation of transcription by light for RCO-1 and RCM-1, the Neurospora homologs of the yeast Tup1-Ssn6 repressor.

The activation of gene transcription by light is transient since light-dependent mRNA accumulation ceases after long exposures to light. This phenomenon, photoadaptation, has been observed in plants and fungi, and allows the perception of changes in light intensities. In the fungus Neurosporacrassa photoadaptation involves the transient binding of the photoresponsive White Collar Complex (WCC) to the promoters of light-regulated genes. We show that RCO-1 and RCM-1, the Neurospora homologs of the components of the yeast Tup1-Ssn6 repressor complex, participate in photoadaptation. Mutation in either rco-1 or rcm-1 result in high and sustained accumulation of mRNAs for con-10 and other light-regulated genes after long exposures to light. The mutation of rco-1 increased the sensitivity to light for con-10 activation and delayed synthesis and/or degradation of con-10 and con-6 mRNAs without altering the amount or the light-dependent phosphorylation of the photoreceptor WC-1. RCO-1 and RCM-1 are located in the Neurospora nuclei were they regulate gene transcription. We show that RCO-1 and RCM-1 participate in the light-transduction pathway of Neurospora and has a role in photoadaptation by repressing gene transcription after long exposures to light.

A complex photoreceptor system mediates the regulation by light of the conidiation genes con-10 and con-6 in Neurospora crassa.

Genes con-10 and con-6 in Neurospora crassa are activated during conidiation or after illumination of vegetative mycelia. Light activation requires the white-collar complex (WCC), a transcription factor complex composed of the photoreceptor WC-1 and its partner WC-2. We have characterized the photoactivation of con-10 and con-6, and we have identified 300bp required for photoactivation in the con-10 promoter. A complex stimulus-response relationship for con-10 and con-6 photoactivation suggested the activity of a complex photoreceptor system. The WCC is the key element for con-10 activation by light, but we suggest that other photoreceptors, the cryptochrome CRY-1, the rhodopsin NOP-1, and the phytochrome PHY-2, modify the activity of the WCC for con-10 photoactivation, presumably through a repressor. In addition we show that the regulatory protein VE-1 is required for full photocarotenogenesis. We propose that these proteins may modulate the WCC in a gene-specific way.

Regulation by blue light of the fluffy gene encoding a major regulator of conidiation in Neurospora crassa.

The development of asexual spores, that is, the process of conidiation, in the fungus Neurospora crassa is increased by light. The fluffy (fl) gene, encoding a major regulator of conidiation, is activated by light. We describe here a detailed characterization of the regulation by blue light of fl in vegetative hyphae. This induction requires the white collar complex (WCC) while the FLD protein acts as a dark repressor of fl transcription. We show that the WCC directly regulates fl transcription in response to blue light after transiently binding the promoter. We propose that fl is repressed by FLD in vegetative mycelia and that the repression is lost after light exposure and WCC activation. The increase in fl mRNA in vegetative mycelia after light exposure, and the corresponding increase in the amount of the regulatory FL protein, should promote the activation of the conidiation pathway. The activation by light of fl provides a simple mechanism for the activation of conidiation by blue light in Neurospora that may be at work in other fungi.

Health and growth of infants breastfed by Norplant contraceptive implants users: a six-year follow-up study.

The objective of the study was to evaluate safety to infants whose mothers used Norplant levonorgestrel implants during breastfeeding. A nonrandomized clinical trial design was used. Participants were 220 and 222 healthy breastfed infants of mothers initiating use of Norplant or T-Cu IUD, respectively, at 55 days to 60 days postpartum. Infants were followed from birth through age 6 years. Breastfeeding pattern, infant growth, and disease events were recorded monthly in the first year, three-monthly in the second, and annually thereafter. Most mothers continued use of Norplant (96.4%) and T-Cu (94.1%) during lactation, and 2140 months of infant exposure to levonorgestrel were accumulated. Breastfeeding pattern and infants growth, from admission through age 6 years, were similar in both groups. In the first year, breastfed infants in the Norplant group had higher incidence rates (p < 0.05) of mild episodes of respiratory infections (adjusted RR 1.17, CI 1.08-1.27), skin conditions (adjusted RR 1.46, CI 1.20-1.79), and eye infections (unadjusted RR 1.49, CI 1.03-2.18) than the control group. Later on, a higher proportion of infants in the T-Cu group showed neurological conditions. Although breastfeeding patterns and infant growth is not affected by Norplant use during lactation, the effect on infants' health of steroidal contraception should be further evaluated.

Amenorrea, anovulación e infertilidad asociadas a la lactancia en un grupo seleccionado de mujeres urbanas / Amenorrhea, anovulation and infertility associated with breast feeding in a selected group of urban women

Se midió la influencia del amamantamiento sobre la duración de la amenorrea en un grupo de mujeres altamente motivadas para amamantar en libre demanda por un tiempo prolongado. Se calculó la probabilidad de presentar el primer sangrado en 676 mujeres en lactancia exclusiva al segundo mes postparto. El 52% presentó el primer sangrado estando en lactancia exclusiva, antes del término del sexto mes postparto. El tiempo transcurrido desde el parto y la suplementación influyeron negativamente en la duración de la amenorrea. Frecuencias de ocho o más mamadas en 24 hooras no lograron prolongar la amenorrea en todos los casos. El reinicio de los ciclos ovulatorios se estudió en 48 mujeres en amenorrea y lactancia exclusiva al tercer mes postparto. El 28% de las mujeres presentó el primer sangrado, y el 26% de los casos ovuló antes del término del sexto mes postparto. La probabilidad acumulada de embarazo fue de 9,4% al sexto mes postparto en las mujeres en lactancia exclusiva que no usaron métodos anticonceptivos. El riesgo fue menor del 2% en aquellos casos que permanecieron en amenorrea durante este período. En la población estudiada la lactancia no resultó un método eficaz para espaciar los nacimientos, con excepción de los casos que se mantuvieron con lactancia exclusiva y amenorrea

Ensayo clínico de anillos vaginales liberadores de progesterona como anticonceptivos en la lactancia / Clinical trial of progesterone releasing vaginal rings as contraceptives during lactation

El objetivo de este ensayo fue investigar los niveles plasmáticos de progesterona y la eficacia anticonceptiva de anillos vaginales liberando progesterona durante la lactancia. Se probaron dos tipos de anillos, que liberaban un promedio aproximado de 5 ó 10 mg. diarios, respectivamente, en 165 mujeres sanas, en plena lactancia. Se constituyó un grupo control de 162 portadores de DIU - T/Cobre. Los anillos fueron insertados el día 60- posparto, y renovados cada tres meses por otros. Al comienzo del ensayo, los niveles plasmáticos de progesterona eran, aproximadamente, 10 nmol/L y 15 nmol/L para anillos que liberaban, respectivamente, 5 y 10 mg por día, y se reducían ligeramente después de 30 días. Los niveles en períodos de uso subsecuentes, observados, se acercaban a los del primer período o etapa. Estos niveles varían dentro de la amplitud eficaz para inhibir la fertilidad de mujeres en lactancia. Se diagnosticó un embarazo en 1005 meses-mujer de uso del anillo de progesterona, y no ocurrió ninguno en 1075 meses-mujer de uso de T/Cu. Esto contrasta con la alta incidencia de embarazos comprobada en un grupo de mujeres en lactancia, sin tratamiento, en quienes se gestaron 19 embarazos, o sea, una incidencia en 677 meses-mujer. No se detectaron efectos deletéreos sobre la lactancia ni sobre el crecimiento de los niños, como tampoco sobre la salud materna o infantil. La conclusión es que el anillo vaginal de progesterona es un método adecuado para el suministro de progesterona como anticonceptivo para madres en lactancia

Influencia de los implantes anticonceptivos Norplant (R) en la lactancia y el crecimiento de los niños / Influence of Norplant contraceptive implants on lactation and infant growth

El presente trabajo estudia la influencia de Norplant (R) sobre la lactancia y sobre el crecimiento del lactante durante el primer año post parto. 100 mujeres en lactancia exclusiva recibieron Norplant (R) el día 55 + ou - 3 post parto y a 100 mujeres con similares características se les insertó una T cobre. No hubo diferencias significativas en la evolución de la lactancia entre los dos grupos hasta el 12§ mes post parto en que el grupo Norplant observó un menor porcentaje de mujeres en lactancia exclusiva. Los niños presentaron un crecimiento ponderal normal en los dos grupos, aunque las hijas del grupo Norplant crecieron significativamente menos durante el 4§ mes de vida, al compararlas con las hijas de mujeres portadoras de DIU. No se diagnosticaron embarazos ni efectos colaterales relevantes. En la leche materna se detectaron concentraciones variables de levonorgestrel con valores que fluctuaron entre 23 y 311 pc/ml. La dosis recibida por el niño se estimó en 15 a 18 ng/Kg/día durante el primer mes de uso de Norplant (R), dosis baja que probablemente no representa riesgo para la salud de los niños, aunque se necesita más investigación sobre el tema. Hasta el momento, el uso de Norplant durante la lactancia debería limitarse a aquellos casos que necesitan protección efectiva y que otros métodos anticonceptivos no hormonales estén contraindicados o no sean aceptados