Significance Early signaling events leading to protection in the heart under cardiac injury are poorly understood. We identified one such protein, A kinase interacting protein (AKIP1), as a modulator ...that responds to oxidative stress; up-regulation of AKIP1 showed protection to ischemic injury through enhanced mitochondrial integrity. We show AKIP1 functions as a molecular scaffold via interaction with mitochondrial apoptosis inducing factor and increases protein kinase A activity. These mitochondrial signaling complexes assembled by AKIP1 alter the physiological response of the heart under ischemic stress. Understanding molecular activity and regulation of AKIP1 could lead to novel therapeutic approaches to limit myocardial injury.
This review summarizes the progress made in our understanding of peroxisome
biogenesis in the last few years, during which the functional roles of many of
the 23 peroxins (proteins involved in ...peroxisomal protein import and peroxisome
biogenesis) have become clearer. Previous reviews in the field have focussed on
the metabolic functions of peroxisomes
(
1
,
2
), aspects of import/biogenesis
(
3
,
4
,
5
,
6
,
7
), the role of peroxins in human
disease (
2
,
8
), and involvement of the endoplasmic
reticulum in peroxisome membrane biogenesis
(
9
,
10
,
11
) as well as the degradation of this
organelle (
5
,
12
). This review refers to some of the
earlier work for the sake of introduction and continuity but deals primarily
with the more recent progress. The principal areas of progress are the
identification of new peroxins, definition of protein-protein interactions
among peroxins leading to the recognition of complexes involved in peroxisomal
protein import, insight into the biogenesis of peroxisomal membrane proteins,
and, of most importance, the elucidation of the role of many conserved peroxins
in human disease. Given the rapid progress in the field, this review also
highlights some of the unanswered questions that remain to be tackled.
Celotno besedilo
Dostopno za:
CMK, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes ...were prepared from wheat (Triticum aestivum). Protein extracts were examined by one‐dimensional and two‐dimensional electrophoresis, followed by high performance liquid chromatography‐tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.
Neural Stem Cells (NSCs) are a distinct group of cells present in the embryonic and adult mammalian central nervous system (CNS) that are able to differentiate into neurons, astrocytes and ...oligodendrocytes. As NSC proliferation declines with age, factors that regulate this process need to be defined. To search for NSC regulatory factors, we performed a quantitative shotgun proteomics study that revealed that members of the High Mobility Group B (HMGB) family are highly expressed in NSCs. Using a neurosphere assay, we report the differential expression of HMGB 1, 2, 3, and 4 mRNAs in proliferating NSCs isolated from various time points during embryonic development, as well as the dynamic expression of HMGB1 and B2 mRNAs and proteins in differentiating embryonic NSCs. Expression of HMGB2 underwent the most dramatic changes during the developmental ages examined; as a result, we assessed its role in NSC proliferation and differentiation. We report the predominance of small diameter HMGB2-/- neurospheres in comparison to wild-type, which correlated with increased proliferation in these smaller HMGB2-/- neurospheres. Our data suggest that HMGB2 plays a regulatory role in NSC cell proliferation and maintenance pathways.
Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein ...kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N6-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr197 and Ser338. Based on its kinetic properties, the engineered C-subunit preferred N6(benzyl)-ATP and N6(phenethyl)-ATP over other ATP analogs, but still retained a 30 μmKm for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.
We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the ...peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor-product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version GFP)-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.
Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions ...in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.
We earlier reported spontaneous features of asthma in Vasoactive Intestinal Peptide knockout mice (VIP KO): 1) peribronchiolar airway inflammation, with accumulation of lymphocytes and eosinophils, ...2) pro-inflammatory cytokine production of IL-5, IL-6, with IFN-γ, and 3) airway hyper-responsiveness to inhaled methacholine. In human asthma, a phenotype with sulfite sensitivity leads to airway inflammation and hyper-responsiveness to inhaled sulfites, and is associated with upregulation of anti-oxidant protein lung carbonyl reductase. For the present experiments, we examined the role of VIP in modulating anti-oxidant genes and their proteins, including lung carbonyl reductase.
Four male VIP KO mice and four wild-type age- and gender matched mice had lungs examined for whole genome microarray and a proteomics approach using mass spectrometry. The proteomics analysis revealed that a novel variant of anti-oxidant protein lung carbonyl reductase (car3) was uniquely and markedly elevated in the VIP KO mice. RT-PCR indicated that carbonic anhydrase 3, which is an anti-oxidant protein, was elevated in the VIP KO mice.
These data support the concept that VIP influences the endogenous oxidant/antioxidant balance. One potential implication is that VIP and its analogues may be used to treat inflammatory diseases, including asthma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumour cells (DTCs) are kept dormant, and what wakes them up, ...are fundamental problems in tumour biology. To address these questions, we used metastasis assays in mice and showed that dormant DTCs reside on microvasculature of lung, bone marrow and brain. We then engineered organotypic microvascular niches to determine whether endothelial cells directly influence breast cancer cell (BCC) growth. These models demonstrated that endothelial-derived thrombospondin-1 induces sustained BCC quiescence. This suppressive cue was lost in sprouting neovasculature; time-lapse analysis showed that sprouting vessels not only permit, but accelerate BCC outgrowth. We confirmed this surprising result in dormancy models and in zebrafish, and identified active TGF-β1 and periostin as tumour-promoting factors derived from endothelial tip cells. Our work reveals that stable microvasculature constitutes a dormant niche, whereas sprouting neovasculature sparks micrometastatic outgrowth.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 ...proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.
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•RNA processing enzymes are enriched near mtDNA nucleoids•SILAC proteomics shows nascent mitochondrial ribosomal proteins with nucleoids•Results suggest cotranscriptional RNA processing and mitoribosome assembly•Mitochondrial nucleoids are sites of RNA processing and ribosome assembly
Mammalian mitochondrial DNA resides in compact nucleoids, where it is replicated and transcribed to generate rRNAs, tRNAs, and mRNAs. Bogenhagen et al. provide insight into the binding events required for mitochondrial ribosome assembly and establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.
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