21 research outputs found
In Vivo Protein Measurements Across Multiple Organs in the Zebrafish
No full textProtein production and degradation are tightly regulated to prevent cellular structures from accumulating damage and to allow their correct functioning. A key aspect of this regulation is the protein half-life, corresponding to the time in which half of a specific protein population is exchanged with respect to its initial state. Proteome-wide techniques to investigate protein half-lives in vivo are emerging. Recently, we have established and thoroughly tested a metabolic labeling approach using C-13 lysine (Lys(6)) for measuring protein lifetimes in mice. The approach is based on the fact that different proteins will incorporate a metabolic label at a rate that is dependent on their half-life. Using amino acid pool modeling and mass spectrometry, it is possible to measure the fraction of newly synthesized proteins and determine protein half-lives. In this chapter, we show how to extend this approach to zebrafish (Danio rerio), using a commercially available fish diet based on the stable isotope labeling by amino acids in cell culture (SILAC) technology. We describe the methods for labeling animals and subsequently use mass spectrometry to determine the lifetimes of a large number of proteins. In the mass spectrometry workflow proposed here, we have implemented the BoxCar data acquisition approach for increasing sample coverage and optimize machine use. To establish the proteome library used in the BoxCar approach, we recommend performing an in-solution digestion followed by peptide fractionation through basic reversed-phase chromatography. Overall, this chapter extends the use of current proteome technologies for the quantification of protein turnover to zebrafish and similar organisms and permits the study of germline changes following specific manipulations
A mass spectrometry workflow for measuring protein turnover rates in vivo.
No full textProteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories
A comprehensive characterization of brain and retina synaptic vesicle proteome by quantitative mass spectrometry.
No full textSynaptic vesicles (SVs) are essential organelles for the transfer of information between a pre-synaptic nerve terminal and a post-synaptic target. SVs release their neurotransmitter content into the synaptic cleft in a tightly regulated process, requiring the orchestrated interaction of a number of proteins involved in exocytosis, endocytosis and vesicular recycling. While the molecular anatomy of brain SV is known, the molecular profile of SVs of sensory systems, such as the retina, is not well understood. The major reason is the unavailability of reliable and efficient isolation protocol for SVs with low amounts of starting material.
In this study, establishment of a modified isolation protocol resulted in successfully purifying highly pure SVs from retina that formed the basis for a reliable determination of its absolute proteome quantification and molecular composition. Remarkably, this protocol allowed recovery of microgram quantities of highly pure and functional SVs from as low as eight bovine retinas. Maximal vesicle recovery was achieved by the introduction of a harsh homogenization step (powdering the tissue in liquid nitrogen using mortar and pestle and subsequent homogenization using Ultra-turrax) followed by subcellular fractionation. Notably, after fractionation by differential and rate-zonal centrifugation, performing an immunoprecipitation with a monoclonal antibody against synatophysin greatly improved the purity and yield of SVs from retina. The purity of the preparation was ensured by western blotting, electron microscopy and mass spectrometry.
The data derived from the iBAQ-MS based absolute quantification of proteins in purified bovine brain and retina SVs from frozen starting materials showed that the copies of SV-integral proteins such as synaptotagmin, SCAMP5, VGlut1 and synaptophysin were similar, if not identical, in bovine brain and retina SVs. Interestingly, however, the copies of v-SNARE protein VAMP2 and tetraspanin protein synaptogyrin-1 were drastically reduced; ~ 6 and 2 fold, respectively, in retina as compared to brain. On the other hand, surprisingly, a three to four fold increase in the copy number of the membrane glycoprotein SV2 were quantified in retina as compared to brain. In addition to differences observed in SV integral proteins, intriguing observations also surfaced when SV associated proteins were quantified. Three copies of the ribbon synapse specific protein syntaxin-3 were found associated to the purified retina SVs, however its brain-specific isoform syntaxin1A/B was totally absent. In addition, the brain-specific Rab3a and synapsin were not quantifiable in our pure retina SVs, suggesting the preparation of SVs to be of majorly ribbon in origin.
These striking differences in the retina SV proteome to that of brain highlights the specialized functionality of retina synapses. Although the molecular profile of synaptic proteins in transverse sections of retina is reported in literature, this is the first study where retina and brain SV proteome have been compared in terms of absolute copies. The findings put forward in this study provides a basis for detailed functional analysis in the future.
In parallel, highly pure rat brain synaptosomes were quantified by iBAQ mass spectrometry in an aim to characterize the average number of presynaptic proteins. As a further validation of the obtained results, a battery of quantitative western blots was run for a selected group of proteins. These results, in combination with additional biophysical and biochemical data, were used to build a three-dimensional model of an average synaptic nerve terminal.
As an independent project, the temporal turnover of synaptic proteins in nerve terminals of the brain and retina were analyzed using a modified version of the SILAC mice approach. To our knowledge this is the first study where a lysine6 diet has been used for labelling proteins over various timeframes to determine protein turnover in vivo. The mice were fed with lysine6 diet for 5, 14 and 21 days and its incorporation in the proteins of brain and retina were analysed by quantitative mass spectrometry. The data shows that the turnover of synaptic proteins in retina is faster than in brain. Strikingly, the turnover of proteins that are involved in similar SV-recycling pathways, correlated well with their respective copy numbers. Future work should be focussed on elucidating the physiological meaning of these interesting observations
Konstribusi PKH Terhadap Kesejahteraan Keluarga Penerima Manfaat (KPM)
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Sunit Agus Tri Cahyono
B2P3KS
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Keywords Effectivity Poverty Prevention Social Capital children underfive commitment elderly empowerment food security incest local culture poverty prevention social capital social conflict social policy social resilience social security social service social welfare street children welfare
Home > Vol 17, No 4 (2018) > Tri Cahyono
Konstribusi PKH Terhadap Kesejahteraan Keluarga Penerima Manfaat (KPM)
Sunit Agus Tri Cahyono
Abstract
Penelitian ini bertujuan mendeskripsikan konstribusi PKH, factor pendukung dan penghambatnya terhadap kesejahteraan Keluarga Penerima Manfaat (KPM) di Kecamatan Candimulyo. Penelitian menggunakan pendekatan kualitatif deskriptif. Wawancara, observasi, dan telaah dokumen digunakan sebagai teknik pengumpulan data. Analisis data menggunakan siklus interaktif. Hasil penelitian menunjukkan, bahwa PKH berkonstribusi kepada peningkatan kesejahteraan keluarga, khususnya mereduksi kekurangan kualitas dan kuantitas pangan, kesehatan, dan pendidikan, sehingga KPM cenderung lebih sejahtera. Factor pendukung antara lain KPM memiliki motivasi/antusiasme tinggi dalam memenuhi komitmen di bidang Pendidikan, kesehatan, dan kesejahteraan social. Faktor penghambat antara lain masih ditemukan eklusif eror dan inklusif eror dalam penetapan sasaran garap (KPM). Rekomendasi yang diajukan diantaranya dalam verifikasi dan validasi (Vefivali) data KPM perlu perluasan pelibatan semua unsur masyarakat mulai dari RT, RW, kelurahan/Desa, kecamatan, kebupaten/kota hingga tokoh masyarakat setempat
A large-scale nanoscopy and biochemistry analysis of postsynaptic dendritic spines
No full textDendritic spines, the postsynaptic compartments of excitatory neurotransmission, have different shapes classified from ‘stubby’ to ‘mushroom-like’. Whereas mushroom spines are essential for adult brain function, stubby spines disappear during brain maturation. It is still unclear whether and how they differ in protein composition. To address this, we combined electron microscopy and quantitative biochemistry with super-resolution microscopy to annotate more than 47,000 spines for more than 100 synaptic targets. Surprisingly, mushroom and stubby spines have similar average protein copy numbers and topologies. However, an analysis of the correlation of each protein to the postsynaptic density mass, used as a marker of synaptic strength, showed substantially more significant results for the mushroom spines. Secretion and trafficking proteins correlated particularly poorly to the strength of stubby spines. This suggests that stubby spines are less likely to adequately respond to dynamic changes in synaptic transmission than mushroom spines, which possibly explains their loss during brain maturation
Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins.
No full textSynaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an "average" synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones
Protein lifetimes in aged brains reveal a proteostatic adaptation linking physiological aging to neurodegeneration
No full textAging is a prominent risk factor for neurodegenerative disorders (NDDs); however, the molecular mechanisms rendering the aged brain particularly susceptible to neurodegeneration remain unclear. Here, we aim to determine the link between physiological aging and NDDs by exploring protein turnover using metabolic labeling and quantitative pulse-SILAC proteomics. By comparing protein lifetimes between physiologically aged and young adult mice, we found that in aged brains protein lifetimes are increased by ~20% and that aging affects distinct pathways linked to NDDs. Specifically, a set of neuroprotective proteins are longer-lived in aged brains, while some mitochondrial proteins linked to neurodegeneration are shorter-lived. Strikingly, we observed a previously unknown alteration in proteostasis that correlates to parsimonious turnover of proteins with high biosynthetic costs, revealing an overall metabolic adaptation that preludes neurodegeneration. Our findings suggest that future therapeutic paradigms, aimed at addressing these metabolic adaptations, might be able to delay NDD onset
Plk1/Polo Phosphorylates Sas-4 at the Onset of Mitosis for an Efficient Recruitment of Pericentriolar Material to Centrosomes
No full textSummary: Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4’s localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion. : Ramani et al. show that Plk1/Polo phosphorylates Drosophila Sas-4 at the onset of mitosis. Cell-cycle-specific modification of Sas-4 determines the spatiotemporal localization of Sas-4 in centrosomes, which is required for an efficient recruitment of PCM proteins in mitosis. Keywords: pericentriolar material, centrosomes, Sas-4, Drosophila melanogaster, Plk1, centrosome maturatio
Contemporary Management of Intracranial Metastatic Disease
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
