Hidden proteome of synaptic vesicles in the mammalian brain

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease

The Coding and Small Non-coding Hippocampal Synaptic RNAome

Neurons are highly compartmentalized cells that depend on local protein synthesis. Messenger RNAs (mRNAs) have thus been detected in neuronal dendrites, and more recently in the pre- and postsynaptic compartments as well. Other RNA species such as microRNAs have also been described at synapses where they are believed to control mRNA availability for local translation. A combined dataset analyzing the synaptic coding and non-coding RNAome via next-generation sequencing approaches is, however, still lacking. Here, we isolate synaptosomes from the hippocampus of young wild-type mice and provide the coding and non-coding synaptic RNAome. These data are complemented by a novel approach for analyzing the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome, and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluidic chamber system, our findings also support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable for studying the dynamics of the synaptic RNAome in response to stimulation. In conclusion, our data provide a valuable resource and point to several important targets for further research

Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm

Abstract The reaction of CO2 with H2O to form bicarbonate (HCO3 −) and H+ controls sperm motility and fertilization via HCO3 −-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3 − in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3 − transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3 − is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3 −/H+ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3 − synthesis. This work provides a new framework for the study of male infertility

Box-and-whisker plot visualization of the percentage change of Total Area Fragment values from peptides between deglycosylated and glycosylated samples in the course of pregnancy and post-partum.

Box-and-whisker plot visualization of the percentage change of Total Area Fragment values from peptides between deglycosylated and glycosylated samples in the course of pregnancy and post-partum.</p

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Fig 4 -

Relative proportions among the different pregnancy states for (a) glycosylated samples and (b) deglycosylated samples. Note the different basic population of Total Area Fragment (a = 4,774,159; b = 7,567,626).</p

Mean percentage change of Total Area Fragment values from boPAGs between deglycosylated (degly) and glycosylated (gly) samples in the course of pregnancy and post-partum.

Mean percentage change of Total Area Fragment values from boPAGs between deglycosylated (degly) and glycosylated (gly) samples in the course of pregnancy and post-partum.</p

Visualization of relative protein abundances measured by Parallel Reaction Monitoring (PRM) mass spectrometry in deglycosylated samples during pregnancy and post-partum.

Visualization of relative protein abundances measured by Parallel Reaction Monitoring (PRM) mass spectrometry in deglycosylated samples during pregnancy and post-partum.</p

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