Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

• Keywords
• integrin heterodimers, integrins, sperm head, α3β1, α6β1, α6β4,
• MeSH
• Models, Biological MeSH
• Integrins chemistry   metabolism   MeSH
• Cell Compartmentation * MeSH
• Protein Multimerization * MeSH
• Mice, Inbred C57BL MeSH
• Protein Subunits metabolism   MeSH
• Protein Domains MeSH
• Spermatozoa metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Integrins MeSH
• Protein Subunits MeSH

Reactive oxygen species (ROS) are essential for life and are involved in the regulation of almost all biological processes. ROS production is critical for plant development, response to abiotic stresses and immune responses. Here, we focus on recent discoveries in ROS biology emphasizing abiotic and biotic stress responses. Recent advancements have resulted in the identification of one of the first sensors for extracellular ROS and highlighted waves of ROS production during stress signalling in Arabidopsis. Enzymes that produce ROS, including NADPH oxidases, exhibit precise regulation through diverse post-translational modifications. Discoveries highlight the importance of both amino- and carboxy-terminal regulation of NADPH oxidases through protein phosphorylation and cysteine oxidation. Here, we discuss advancements in ROS compartmentalization, systemic ROS waves, ROS sensing and post-translational modification of ROS-producing enzymes and identify areas where foundational gaps remain.

• MeSH
• Arabidopsis enzymology   physiology   MeSH
• Stress, Physiological * MeSH
• Reactive Oxygen Species metabolism   MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Reactive Oxygen Species MeSH

The ribosome, owing to its exceptional conservation, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt 'big' protoribosome model, which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilized by the peptides. In contrast, the 136nt 'small' model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the 'small' protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA-protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA-protein interactions crucial for the ribosomal structural integrity.

• MeSH
• Nucleic Acid Conformation MeSH
• Models, Molecular MeSH
• Peptides chemistry   metabolism   MeSH
• Peptidyl Transferases metabolism   chemistry   MeSH
• Ribosomal Proteins * metabolism   chemistry   MeSH
• Ribosomes * metabolism   MeSH
• RNA metabolism   chemistry   MeSH
• RNA Stability MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides MeSH
• Peptidyl Transferases MeSH
• Ribosomal Proteins * MeSH
• RNA MeSH

One strategy to control the morphology of hybrid polymeric nanostructures is the proper selection of macromolecule architecture. We prepared metallacarborane-rich nanoparticles by interaction of double-hydrophilic block copolymers consisting of both poly(2-alkyl oxazolines) and poly(ethylene oxide) blocks with cobaltabisdicarbollide anion in physiological saline. The inner structure of the hybrid nanoparticles was studied by cryo-TEM, light scattering, SAXS, NMR, and ITC. Although the thermodynamics of diblock and star-like systems are almost identical, the macromolecular architecture has a great impact on the size and inner morphology of the nanoparticles. While hybrid nanoparticles formed by linear diblock copolymers are homogeneous, resembling gel-like nanospheres, the star-like shape of 4-arm block copolymers with PEO blocks in central parts of macromolecules leads to distinct compartmentalization. Because metallacarboranes are promising species in medicine, the studied nanoparticles are important for targeted drug delivery of boron cluster compounds.

• Publication type
• Journal Article MeSH

Allelochemicals play important roles in the plant defense against herbivorous insects. They act as feeding deterrents, interfere with digestion and nutrient absorption, and cause production of potentially dangerous oxidative radicals. This study demonstrates that the distributions of oxidative radicals and of the antioxidant enzymes that eliminate them are compartmentalized in the digestive tract of Spodoptera littoralis larvae. Feeding on diets supplemented with the tannic acid (TA), alpha-solanine, and demissidine, respectively, did not affect the rate of food passage through the digestive tract of larvae but 1.25, 2.5, and 5% TA evoked a strong oxidative response. The amount of the superoxide anion in the foregut tissue and content increased up to 70-fold and the titer of total peroxides in the foregut content about 3-fold. This oxidative stress was associated with enhanced carbonyl content in the foregut tissue proteins, indicative of certain tissue deterioration. Extensive foregut damage was probably prevented by elevated activity of the glutathione S-transferase peroxidase. A complex antioxidant response was elicited in the midgut. The activities of superoxide dismutase and catalase increased significantly in the midgut tissue and content, and the activity of ascorbate peroxidase rose in the midgut tissue. The enzymes apparently eliminated oxidative radicals passing to midgut from the foregut with the food bolus and thereby prevented carbonylation of the midgut proteins. We postulate that the generation of oxidative radicals in the foregut and the induction of antioxidant defense in the midgut are controlled processes and that their compartmentalization is an important functional feature of the digestive tract. The glycoalkaloid alpha-solanine and the aglycone demissidine applied at 0.05 and 0.1% concentrations had no effect on any of the examined parameters.

• MeSH
• Solanaceous Alkaloids metabolism   MeSH
• Ascorbate Peroxidases MeSH
• Glutathione Peroxidase metabolism   MeSH
• Catalase metabolism   MeSH
• Larva metabolism   MeSH
• Oxidative Stress physiology   MeSH
• Peroxidases metabolism   MeSH
• Solanine metabolism   MeSH
• Spodoptera enzymology   metabolism   MeSH
• Superoxide Dismutase metabolism   MeSH
• Superoxides metabolism   MeSH
• Tannins metabolism   MeSH
• Digestive System metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Solanaceous Alkaloids MeSH
• Ascorbate Peroxidases MeSH
• demissidine MeSH Browser
• Glutathione Peroxidase MeSH
• Catalase MeSH
• Peroxidases MeSH
• Solanine MeSH
• Superoxide Dismutase MeSH
• Superoxides MeSH
• Tannins MeSH

The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.

• Keywords
• BBSome, MKS/B9 complex, cilium/flagellum, transition zone, trypanosome,
• MeSH
• Bardet-Biedl Syndrome genetics   metabolism   MeSH
• Basal Bodies metabolism   ultrastructure   MeSH
• Cilia genetics   metabolism   MeSH
• Ciliopathies genetics   metabolism   MeSH
• Cytoskeleton metabolism   ultrastructure   MeSH
• Encephalocele genetics   metabolism   MeSH
• Flagella genetics   metabolism   ultrastructure   MeSH
• Microscopy, Fluorescence MeSH
• Cell Compartmentation MeSH
• Humans MeSH
• Mutation MeSH
• Polycystic Kidney Diseases genetics   metabolism   MeSH
• Ciliary Motility Disorders genetics   metabolism   MeSH
• Proteome genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Retinitis Pigmentosa MeSH
• RNA Interference MeSH
• Microscopy, Electron, Transmission MeSH
• Trypanosoma genetics   metabolism   ultrastructure   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH
• Protozoan Proteins MeSH

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

• MeSH
• Cell Membrane metabolism   MeSH
• Sperm Capacitation * MeSH
• Swine MeSH
• Proteasome Endopeptidase Complex metabolism   MeSH
• Proteomics MeSH
• Spermatozoa cytology   physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• ATP dependent 26S protease MeSH Browser
• Proteasome Endopeptidase Complex MeSH

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described "heavy rafts" or non-raft membrane. Our data show that only Csk targeted into "classical" raft but not to "heavy raft" or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.

• MeSH
• Cell Membrane metabolism   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Phosphorylation MeSH
• Immunoblotting MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Membrane Microdomains * MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• Receptors, Antigen, T-Cell metabolism   MeSH
• Signal Transduction MeSH
• src-Family Kinases MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CSK Tyrosine-Protein Kinase MeSH
• CSK protein, human MeSH Browser
• Proto-Oncogene Proteins MeSH
• Receptors, Antigen, T-Cell MeSH
• src-Family Kinases MeSH
• Protein-Tyrosine Kinases MeSH

Trastuzumab is effective in about half of HER2-positive breast cancer patients. The PI3K/Akt signalling pathway plays an important role in the process of primary and secondary resistance to anti-HER2 targeted therapy. We evaluated the relationship between expression, activation and subcellular localization of selected Akt isoforms and response to trastuzumab-based anti-HER2 targeted therapy in patients with HER2-positive metastatic breast cancer. Seventy-four women with verified HER2-positive breast cancer were treated with trastuzumab for metastatic disease. Immunohistochemistry was used to evaluate Akt1, Akt2, pAkt Thr308 and pAkt Ser473 expression. For pAkt, cytoplasmic and nuclear fractions were assessed separately. Even though Akt isoforms were expressed in the majority of tumours, activated Akt (pAkt) was present in the cytoplasm only and not in the nucleus in >20% of tumours, and there was no pAkt at all in another 7-13% of tumours. Patients whose tumours showed strong Akt2 expression and had pAkt (pAkt-Thr308 and/or pAkt-Ser473) detectable in the cytoplasm as well as nucleus (n+c), exhibited improved time to progression (TTP) and overall survival from the initiation of trastuzumab therapy (OSt). Patients with tumours with strong Akt2 and pAkt Thr308 (n+c) had superior TTP (17.0 vs. 7.6 months, P=0.024; HR 0.52) and OSt (51.8 vs. 16.8 months, P=0.0009; HR 0.34) compared to other tumours. Similar results were found for strong Akt2 and pAkt Ser473 (n+c): TTP 13.1 vs. 7.2 months (P=0.085, HR 0.62) and OSt 50.8 vs. 17.0 months (P=0.009; HR 0.45). This study is the first to prove the significance of Akt kinase isoform, activity and compartmentalization for the prediction of response to trastuzumab-based therapy in patients with HER2-positive metastatic breast cancer.

• MeSH
• Adult MeSH
• Antibodies, Monoclonal, Humanized administration & dosage   MeSH
• Cell Compartmentation MeSH
• Middle Aged MeSH
• Humans MeSH
• Neoplasm Metastasis drug therapy   genetics   pathology   MeSH
• Breast Neoplasms drug therapy   genetics   pathology   MeSH
• Prognosis MeSH
• Proto-Oncogene Proteins c-akt biosynthesis   MeSH
• Receptor, ErbB-2 MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Aged MeSH
• Signal Transduction drug effects   MeSH
• Neoplasm Staging MeSH
• Trastuzumab MeSH
• Treatment Outcome MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ERBB2 protein, human MeSH Browser
• Antibodies, Monoclonal, Humanized MeSH
• Proto-Oncogene Proteins c-akt MeSH
• Receptor, ErbB-2 MeSH
• Trastuzumab MeSH

Nuclear phosphoinositides are recognized as regulators of many nuclear processes including chromatin remodeling, splicing, transcription, DNA repair and epigenetics. These processes are spatially organized in different nuclear compartments. Phase separation is involved in the formation of various nuclear compartments and molecular condensates separated from surrounding environment. The surface of such structures spatiotemporally coordinates formation of protein complexes. PI(4,5)P2 (PIP2) integration into phase-separated structures might provide an additional step in their spatial diversification by attracting certain proteins with affinity to PIP2. Our laboratory has recently identified novel membrane-free PIP2-containing structures, so called Nuclear Lipid Islets (NLIs). We provide an evidence that these structures are evolutionary conserved in different organisms. We hypothesize that NLIs serve as a scaffolding platform which facilitates the formation of transcription factories, thus participating in the formation of nuclear architecture competent for transcription. In this review we speculate on a possible role of NLIs in the integration of various processes linked to RNAPII transcription, chromatin remodeling, actin-myosin interaction, alternative splicing and lamin structures.

• Keywords
• Nuclear architecture, Nucleus, Phase separation, Phosphoinositides, Transcription,
• MeSH
• Chromatin genetics   metabolism   MeSH
• Phosphatidylinositol 4,5-Diphosphate genetics   metabolism   MeSH
• Humans MeSH
• Chromatin Assembly and Disassembly * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Chromatin MeSH
• Phosphatidylinositol 4,5-Diphosphate MeSH