Functional interdependence of the actin regulators CAP1 and cofilin1 in control of dendritic spine morphology

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are both crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Because actin filaments (F-actin) are the major structural component in spines, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Indeed, mouse studies identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission and behavior. These studies emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. We report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperated with cofilin1 in spines and that its helical folded domain mediated this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that was essential for synaptic cofilin1 activity.

The Functional Role of Extracellular Matrix Proteins in Cancer

The extracellular matrix (ECM) is highly dynamic as it is constantly deposited, remodeled and degraded to maintain tissue homeostasis. ECM is a major structural component of the tumor microenvironment, and cancer development and progression require its extensive reorganization. Cancerized ECM is biochemically different in its composition and is stiffer compared to normal ECM. The abnormal ECM affects cancer progression by directly promoting cell proliferation, survival, migration and differentiation. The restructured extracellular matrix and its degradation fragments (matrikines) also modulate the signaling cascades mediated by the interaction with cell-surface receptors, deregulate the stromal cell behavior and lead to emergence of an oncogenic microenvironment. Here, we summarize the current state of understanding how the composition and structure of ECM changes during cancer progression. We also describe the functional role of key proteins, especially tenascin C and fibronectin, and signaling molecules involved in the formation of the tumor microenvironment, as well as the signaling pathways that they activate in cancer cells.

PBP1 of Staphylococcus aureus has multiple essential functions in cell division

Bacterial cell division is a complex process requiring the coordination of multiple components, to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multi–stage PG architecture that develops during septation. Penicillin binding proteins (PBPs) are essential for the final steps of PG biosynthesis — their transpeptidase activity links together the peptide sidechain of nascent glycan strands together. PBP1 is required for cell division in S. aureus and here we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C–terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thus coordinate the cell division process. The transpeptidase activity of PBP1 is also essential but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1–specific β–lactam, meropenem. Together these results lead to a model for septal PG synthesis where PBP1 enzyme activity is responsible for the characteristic architecture of the septum and PBP1 protein molecules coordinate cell division allowing septal plate formation.

Ancient origin of fucosylated xyloglucan in charophycean green algae

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.

Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs in vitro and consequently can alter the proteome of NETs. However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) in vitro. Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.

Heterozygous lamin B1 and lamin B2 variants cause primary microcephaly and define a novel laminopathy

Purpose Lamins are the major component of nuclear lamina, maintaining structural integrity of the nucleus. Lamin A/C variants are well established to cause a spectrum of disorders ranging from myopathies to progeria, termed laminopathies. Phenotypes resulting from variants in LMNB1 and LMNB2 have been much less clearly defined. Methods We investigated exome and genome sequencing from the Deciphering Developmental Disorders Study and the 100,000 Genomes Project to identify novel microcephaly genes. Results Starting from a cohort of patients with extreme microcephaly, 13 individuals with heterozygous variants in the two human B-type lamins were identified. Recurrent variants were established to be de novo in nine cases and shown to affect highly conserved residues within the lamin ɑ-helical rod domain, likely disrupting interactions required for higher-order assembly of lamin filaments. Conclusion We identify dominant pathogenic variants in LMNB1 and LMNB2 as a genetic cause of primary microcephaly, implicating a major structural component of the nuclear envelope in its etiology and defining a new form of laminopathy. The distinct nature of this lamin B–associated phenotype highlights the strikingly different developmental requirements for lamin paralogs and suggests a novel mechanism for primary microcephaly warranting future investigation.

Synthetic cell-permeable caveolin-1 scaffolding domain peptide activates phagocytosis of Escherichia coli by regulating Rab5 activity

Caveolae are defined as 50–100 nm wide pits in the plasma membrane containing oligomeric caveolin proteins. They have been implicated in endocytosis (including phagocytosis), transcytosis, calcium signalling, and numerous other signal transduction events. Caveolin-1, a major structural component of caveolae, enhances Rab5 activity. In this study, we examined the effect of a synthetic cell-permeable peptide of the caveolin-1 scaffolding domain (CSD) on phagocytosis. Treatment with the CSD peptide increased Rab5 activity, Rab5-early endosome antigen 1 (EEA1) interaction, and phagocytosis of Escherichia coli. The results suggest that the synthetic cell-permeable CSD peptide is an activator of phagocytosis.

STUDY OF CHITINASE AND CHITINOLYTIC ACTIVITY OF LACTOBACILLUS STRAINS

The increasing consumer demand for less processed and more natural food products – while improving those products’ quality, safety, and shelf-life – has raised the necessity of chemical preservative replacement. Biopreservation refers to extended storage life and enhanced safety of foods using the natural microflora and (or) their antibacterial products. Chitinolytic enzymes are of biotechnological interest, since their substrate, chitin, is a major structural component of the cell wall of fungi, which are the main cause of the spoilage of food and raw plant material. Among the several organisms, many bacteria produce chitinolytic enzymes, however, this behaviour is not general. The chitinase activity of the lactic acid bacteria is scarcely known and studied.The aim of the present study was to select Lactobacillus strains that have genes encoding chitinase, furthermore, to detect expressed enzymes and to characterise their chitinase activity. Taking into consideration the importance of chitin-bindig proteins (CBPs) in the chitinase activity, CBPs were also examined. Five Lactobacillus strains out of 43 strains from 12 different species were selected by their chitinase coding gene. The presence of the chitinase and chitin-biding protein production were confirmed, however, no chitinolytic activity has been identified.

Shear Stress Regulation of Endothelial Glycocalyx Structure Is Determined by Glucobiosynthesis

Objective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with 13 C-labeled glucose. Conclusions: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.

Movement of accessible plasma membrane cholesterol by the GRAMD1 lipid transfer protein complex

Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.