Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.     

The CORVET Subunit Vps8 Cooperates with the Rab5 Homolog Vps21 to Induce Clustering of Late Endosomal Compartments

Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.     

Effects of a lipid environment on the fibrillogenic pathway of the N-terminal polypeptide of human apolipoprotein A-I, responsible for in vivo amyloid fibril formation

In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an α-helical state, which undergoes aggregation at 37°C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of α-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.     

Mechanical properties of zona pellucida hardening

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP.     

An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis

Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.     

Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.     

Atg9 reservoirs, a new organelle of the yeast endomembrane system?

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.     

The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.     

Phylogeographic analysis of haplogroup E3b (E-M215) y chromosomes reveals multiple migratory events within and out of Africa

We explored the phylogeography of human Y-chromosomal haplogroup E3b by analyzing 3401 individuals from five continents. Our data refine the phylogeny of the entire haplogroup, which appears as a collection of lineages with very different evolutionary histories, and reveal signatures of several distinct processes of migrations and/or recurrent gene flow that occurred in Africa and western Eurasia over the past 25000 years. In Europe, the overall frequency pattern of haplogroup E-M78 does not support the hypothesis of a uniform spread of people from a single parental Near Eastern population. The distribution of E-M81 chromosomes in Africa closely matches the present area of distribution of Berber-speaking populations on the continent, suggesting a close haplogroup-ethnic group parallelism. E-M34 chromosomes were more likely introduced in Ethiopia from the Near East. In conclusion, the present study shows that earlier work based on fewer Y-chromosome markers led to rather simple historical interpretations and highlights the fact that many population-genetic analyses are not robust to a poorly resolved phylogeny.     

The molecular dissection of mtDNA haplogroup H confirms that the Franco-Cantabrian glacial refuge was a major source for the European gene pool

Complete sequencing of 62 mitochondrial DNAs (mtDNAs) belonging (or very closely related) to haplogroup H revealed that this mtDNA haplogroup--by far the most common in Europe--is subdivided into numerous subhaplogroups, with at least 15 of them (H1-H15) identifiable by characteristic mutations. All the haplogroup H mtDNAs found in 5,743 subjects from 43 populations were then screened for diagnostic markers of subhaplogroups H1 and H3. This survey showed that both subhaplogroups display frequency peaks, centered in Iberia and surrounding areas, with distributions declining toward the northeast and southeast--a pattern extremely similar to that previously reported for mtDNA haplogroup V. Furthermore, the coalescence ages of H1 and H3 (~11,000 years) are close to that previously reported for V. These findings have major implications for the origin of Europeans, since they attest that the Franco-Cantabrian refuge area was indeed the source of late-glacial expansions of hunter-gatherers that repopulated much of Central and Northern Europe from ~15,000 years ago. This has also some implications for disease studies. For instance, the high occurrence of H1 and H3 in Iberia led us to re-evaluate the haplogroup distribution in 50 Spanish families affected by nonsyndromic sensorineural deafness due to the A1555G mutation. The survey revealed that the previously reported excess of H among these families is caused entirely by H3 and is due to a major, probably nonrecent, founder event.