Genetic constraints on protein evolution

Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

Self-assembly of human latexin into amyloid-like oligomers

Background  

In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations.     

Loss of hepatic chaperone‐mediated autophagy accelerates proteostasis failure in aging

Chaperone‐mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver‐specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.     

Saturated fat intake and alcohol consumption modulate the association between the APOE polymorphism and risk of future coronary heart disease: a nested case-control study in the Spanish EPIC cohort

The association is still not clear between the common APOE polymorphism and coronary heart disease (CHD) risk, nor its modulation by diet. Thus, our aim was to study the association between the APOE genotypes and incident CHD and how dietary fat and alcohol consumption modify these effects. We performed a nested case-control study in the Spanish European Prospective Investigation into Cancer and Nutrition cohort. Healthy men and women (41?440, 30-69 years) were followed up over a 10-year period, with the incident CHD cases being identified. We analyzed 534 incident CHD cases and 1123 controls. APOE, dietary intake and plasma lipids were determined at baseline. The APOE polymorphism was significantly associated with low-density lipoprotein cholesterol (LDL-C), and gene-alcohol interactions in determining LDL-C were detected. In the whole population, the E2 allele was significantly associated with a lower CHD risk than E3/E3 subjects [odds ratio (OR), 0.58; 95% confidence interval (CI), 0.38-0.89]. The E4 allele did not reach statistical significance vs. E3/E3 (OR, 1.17; 95% CI, 0.88-1.58). However, saturated fat intake modified the effect of the APOE polymorphism in determining CHD risk. When saturated fat intake was low (<10% of energy), no statistically significant association between the APOE polymorphism and CHD risk was observed (P=.682). However, with higher intake (≥10%), the polymorphism was significant (P=.005), and the differences between E2 and E4 carriers were magnified (OR for E4 vs. E2, 3.33; 95% CI, 1.61-6.90). Alcohol consumption also modified the effect of the APOE on CHD risk.In conclusion, in this Mediterranean population, the E2 allele is associated with lower CHD risk, and this association is modulated by saturated fat and alcohol consumption.     

Dynamics and mechanical stability of the developing dorsoventral organizer of the wing imaginal disc

Shaping the primordia during development relies on forces and mechanisms able to control cell segregation. In the imaginal discs of Drosophila the cellular populations that will give rise to the dorsal and ventral parts on the wing blade are segregated and do not intermingle. A cellular population that becomes specified by the boundary of the dorsal and ventral cellular domains, the so-called organizer, controls this process. In this paper we study the dynamics and stability of the dorsal-ventral organizer of the wing imaginal disc of Drosophila as cell proliferation advances. Our approach is based on a vertex model to perform in silico experiments that are fully dynamical and take into account the available experimental data such as: cell packing properties, orientation of the cellular divisions, response upon membrane ablation, and robustness to mechanical perturbations induced by fast growing clones. Our results shed light on the complex interplay between the cytoskeleton mechanics, the cell cycle, the cell growth, and the cellular interactions in order to shape the dorsal-ventral organizer as a robust source of positional information and a lineage controller. Specifically, we elucidate the necessary and sufficient ingredients that enforce its functionality: distinctive mechanical properties, including increased tension, longer cell cycle duration, and a cleavage criterion that satisfies the Hertwig rule. Our results provide novel insights into the developmental mechanisms that drive the dynamics of the DV organizer and set a definition of the so-called Notch fence model in quantitative terms.     

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.     

The active human gut microbiota differs from the total microbiota

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around 75% of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis.Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.     

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.