Disaggregating chaperones: an unfolding story.

Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Here, we review the role of molecular chaperones as a network of cellular defenses against the formation of cytotoxic protein aggregates. Chaperones favour the native folding of proteins either as "holdases", sequestering hydrophobic regions in misfolding polypeptides, and/or as "unfoldases", forcibly unfolding and disentangling misfolded polypeptides from aggregates. Whereas in bacteria, plants and fungi Hsp70/40 acts in concert with the Hsp100 (ClpB) unfoldase, Hsp70/40 is the only known chaperone in the cytoplasm of mammalian cells that can forcibly unfold and neutralize cytotoxic protein conformers. Owing to its particular spatial configuration, the bulky 70 kDa Hsp70 molecule, when distally bound through a very tight molecular clamp onto a 50-fold smaller hydrophobic peptide loop extruding from an aggregate, can locally exert on the misfolded segment an unfolding force of entropic origin, thus destroying the misfolded structures that stabilize aggregates. ADP/ATP exchange triggers Hsp70 dissociation from the ensuing enlarged unfolded peptide loop, which is then allowed to spontaneously refold into a closer-to-native conformation devoid of affinity for the chaperone. Driven by ATP, the cooperative action of Hsp70 and its co-chaperone Hsp40 may thus gradually convert toxic misfolded protein substrates with high affinity for the chaperone, into non-toxic, natively refolded, low-affinity products. Stress- and mutation-induced protein damages in the cell, causing degenerative diseases and aging, may thus be effectively counteracted by a powerful network of molecular chaperones and of chaperone-related proteases.

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Cellular source and mechanisms of high transcriptome complexity in the Mammalian testis.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.