Progressive alterations of striatal polysomal architecture in a mouse model of Huntington's disease

Neurons rely on a precise spatial and temporal control of protein synthesis due to their highly polarized morphology and their functional singularities. Consequently, alterations in protein translation have been widely related to the development and progression of various neurological and neurodegenerative disorders, including Huntington's disease. Here we explored the architecture of polysomes in their native brain context by performing 3D electron tomography of striatal tissue derived from a knock-in mouse model of the disease. Results showed a progressive remodelling towards a polysomal compacted architecture that parallels in time the emergence and progression of symptoms in the mouse model. The aberrant architecture is compatible with ribosome stalling phenomena and, in fact, we detected an increase in the expression of the stalling release factor eIF5A2. Polysomal sedimentation gradients showed significant excess in the accumulation of free 40S ribosomal subunits in heterozygous striatal samples. Overall the results indicate that changes in the architecture of the protein synthesis machinery might be at the basis of translational alterations associated to Huntington's disease and open new avenues for understanding disease progression.

WWOX Regulates UV/Cold Shock-Mediated Calcium Influx and Nuclear Bubbling in Frostbite

UV/cold shock-mediated frostbite involves non-apoptotic nuclear bubbling cell death (BCD) and participation of functional WWOX in cells (WWOXf). In contrast, cells with WWOX deficiency or dysfunction (WWOXd) undergo pop-out explosion death (POD). Here, by time-lapse microscopy, when WWOXf cells were exposed to UV or UV/cold shock and then incubated at room temperature, these cells rapidly and sequentially underwent: 1) loss of mitochondrial membrane potential, 2) formation of a nitric oxide (NO)-containing nuclear bubble per cell, 3) WWOX-dependent increase in calcium (Ca2+) influx, 4) shutdown of mRNA and protein synthesis machinery, as determined by RT/PCR and gene chip analysis, and 5) eventual cell death without caspase activation, stress fiber formation and chromosomal DNA fragmentation. In contrast, WWOXd cells exhibited a faster kinetics of stress fiber formation, explosion and death without NO production. Ectopic WWOX restored calcium influx and nuclear bubbling in WWOXd cells. In hairless mice, UV/cold shock rapidly downregulated protein expression in the skin and then liver, which may lead to organ damages. UV/cold shock induced complex formation of antiapoptotic TRAF2 and proapoptotic WWOX and their co-translocation to the nucleus, where the complex dissociation occurred. The observations suggest that WWOX and TRAF2 dissociation is needed for nuclear bubbling and death.

Active Translation Control of CD4 T Cell Activation by Regulatory T Cells

Increased protein synthesis is a hallmark of lymphocyte activation. Regulatory T cells (Tregs) suppress the activation and subsequent effector functions of CD4 effector T cells (Teff). Molecular mechanisms that enforce suppression on CD4 Teff cell function are unclear. Control of CD4 Teff cell activation by Tregs has largely been defined at the transcriptional level, which does not reflect changes in post-transcriptional control. We found that Tregs suppressed activation-induced global protein synthesis in CD4 Teff cells prior to cell division. We analyzed genome-wide changes in the transcriptome and translatome of activated CD4 Teff cells using two independent approaches. We show that mRNAs encoding for the protein synthesis machinery are regulated at the level of translation in activated Teff cells. Strikingly, Tregs suppressed global protein synthesis of CD4 Teff cells by specifically inhibiting mRNAs of the translation machinery at the level of mTORC1-mediated translation control. Lastly, we found that the RNA helicase eIF4A inhibitor rocaglamide A (RocA) can suppress CD4 Teff activation in vitro to alleviate inflammatory CD4 Teff activation caused by acute Treg depletion in vivo. These data provide evidence that peripheral tolerance is enforced by Tregs, mediated by IL-10, through mRNA translational control in CD4 Teff cells. Therefore, therapeutic targeting of the protein synthesis machinery can mitigate inflammatory responses invoked by Treg loss of function.

Reduced Lytic Activity of Bacteriophages in Presence of Antibiotics Targeting Bacterial Protein Synthesis

Combination therapy of bacteriophage and antibiotics offers promise to treat multiple drug resistant bacterial infections through phage antibiotic synergy. However, its usage requires careful assessment as most antibiotics with mechanisms dependent upon inhibiting cell growth through interfering bacterial protein synthesis machinery were found to have an antagonistic effect on phage activity.

Broad Influence of Mutant Ataxin-3 on the Proteome of the Adult Brain, Young Neurons, and Axons Reveals Central Molecular Processes and Biomarkers in SCA3/MJD Using Knock-In Mouse Model

Spinocerebellar ataxia type 3 (SCA3/MJD) is caused by CAG expansion mutation resulting in a long polyQ domain in mutant ataxin-3. The mutant protein is a special type of protease, deubiquitinase, which may indicate its prominent impact on the regulation of cellular proteins levels and activity. Yet, the global model picture of SCA3 disease progression on the protein level, molecular pathways in the brain, and neurons, is largely unknown. Here, we investigated the molecular SCA3 mechanism using an interdisciplinary research paradigm combining behavioral and molecular aspects of SCA3 in the knock-in ki91 model. We used the behavior, brain magnetic resonance imaging (MRI) and brain tissue examination to correlate the disease stages with brain proteomics, precise axonal proteomics, neuronal energy recordings, and labeling of vesicles. We have demonstrated that altered metabolic and mitochondrial proteins in the brain and the lack of weight gain in Ki91 SCA3/MJD mice is reflected by the failure of energy metabolism recorded in neonatal SCA3 cerebellar neurons. We have determined that further, during disease progression, proteins responsible for metabolism, cytoskeletal architecture, vesicular, and axonal transport are disturbed, revealing axons as one of the essential cell compartments in SCA3 pathogenesis. Therefore we focus on SCA3 pathogenesis in axonal and somatodendritic compartments revealing highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport and mitochondria was decreased. We demonstrate the accumulation of axonal vesicles in neonatal SCA3 cerebellar neurons and increased phosphorylation of SMI-312 positive adult cerebellar axons, which indicate axonal dysfunction in SCA3. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of various components of protein synthesis machinery in axons.

Mechanistic studies of non-canonical amino acid mutagenesis

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

Adaptive immunity at the crossroads of autophagy and metabolism

The function of lymphocytes is dependent on their plasticity, particularly their adaptation to energy availability and environmental stress, and their protein synthesis machinery. Lymphocytes are constantly under metabolic stress, and macroautophagy/autophagy is the primary metabolic pathway that helps cells overcome stressors. The intrinsic role of autophagy in regulating the metabolism of adaptive immune cells has recently gained increasing attention. In this review, we summarize and discuss the versatile roles of autophagy in regulating cellular metabolism and the implications of autophagy for immune cell function and fate, especially for T and B lymphocytes.