Resolving the zinc binding capacity of honey bee vitellogenin and locating its putative binding sites.

The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg-laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc-carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc-binding by this protein are not well-understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+ -ions. Our full-length protein structure and sequence analysis revealed seven potential zinc-binding sites. These are located in the ß-barrel and α-helical subdomains of the N-terminal domain, the lipid binding site, and the cysteine-rich C-terminal region of unknown function. Interestingly, two potential zinc-binding sites in the ß-barrel can support a proposed role for this structure in DNA-binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+ -ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg-laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers.

Binding Specificity of ASHH2 CW Domain Toward H3K4me1 Ligand Is Coupled to Its Structural Stability Through Its α1-Helix.

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site-the two tryptophans and the α1-helix form and maintain the binding pocket- were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.

Functional tests in patients with ischemic heart disease.

Lately, easier and shorter tests have been used in the functional evaluation of cardiac patients. Among these, walking speed (WS) and Timed Up and Go (TUG) tests are associated with all-cause mortality, mainly cardiovascular and the rate of re-hospitalization, especially in the elderly population. We prospectively analyzed a group of 38 patients admitted to the Cardiology Clinic from Elias Hospital, Romania, with chronic coronary syndrome (CCS) (n=22) and STEMI (n=16). We assessed the patients immediately after admission and before discharge with G-WALK between the 1st and 30th of September 2019. Our study group had a mean age of 62.7±12.1 years. Patients with a low WS were older (69.90±12.84 vs. 59.90±10.32 years, p=0.02) and had a lower serum hemoglobin (12.38±1.20 vs. 13.72±2.07 g/dl, p=0.02). The WS significantly improved during hospitalization (p=0.03) after optimal treatment. The TUG test performed at the time of admission had a longer duration in patients with heart failure (14.05 vs. 10.80 sec, p=0.02) and was influenced by patients' age (r=0.567, p=0.02), serum creatinine (r=0.409, p=0.03) and dilation of right heart chambers (r=0.399, p=0.03). WS and TUG tests can be used in patients with CCS and STEMI, and are mainly influenced by age, thus having a greater value among the elderly.

Phosphoinositide 3-kinase signalling in the nucleolus.

The phosphoinositide 3-kinase (PI3K) signalling pathway plays key roles in many cellular processes and is altered in many diseases. The function and mode of action of the pathway have mostly been elucidated in the cytoplasm. However, many of the components of the PI3K pathway are also present in the nucleus at specific sub-nuclear sites including nuclear speckles, nuclear lipid islets and the nucleolus. Nucleoli are membrane-less subnuclear structures where ribosome biogenesis occurs. Processes leading to ribosome biogenesis are tightly regulated to maintain protein translation capacity of cells. This review focuses on nucleolar PI3K signalling and how it regulates rRNA synthesis, as well as on the identification of downstream phosphatidylinositol (3,4,5)trisphosphate effector proteins.

Nuclear Phosphatidylinositol 3,4,5-Trisphosphate Interactome Uncovers an Enrichment in Nucleolar Proteins.

Polyphosphoinositides (PPIns) play essential roles as lipid signaling molecules, and many of their functions have been elucidated in the cytoplasm. However, PPIns are also intranuclear where they contribute to chromatin remodeling, transcription, and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), has been mapped to the nucleus and nucleoli, but its role remains unclear in this subcellular compartment. To gain further insights into the nuclear functions of PtdIns(3,4,5)P3, we applied a previously developed quantitative MS-based approach to identify the targets of PtdIns(3,4,5)P3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5)P3-interacting partners, and gene ontology analysis for the biological functions of this dataset revealed an enrichment in RNA processing/splicing, cytokinesis, protein folding, and DNA repair. Interestingly, about half of these interactors were common to nucleolar protein datasets, some of which had dual functions in rRNA processes and DNA repair, including poly(ADP-ribose) polymerase 1 (PARP1, now referred as ADP-ribosyltransferase 1). PARP1 was found to interact directly with PPIn via three polybasic regions in the DNA-binding domain and the linker located N-terminal of the catalytic region. PARP1 was shown to bind to PtdIns(3,4,5)P3 as well as phosphatidylinositol 3,4-bisphosphate in vitro and to colocalize with PtdIns(3,4,5)P3 in the nucleolus and with phosphatidylinositol 3,4-bisphosphate in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5)P3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5)P3-mediated interactions in the nucleus and nucleolus.

Nuclear upregulation of class I phosphoinositide 3-kinase p110ß correlates with high 47S rRNA levels in cancer cells.

The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110ß are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110ß) is seldom mutated compared with PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are, however, unclear. We have previously reported that p110ß is highly expressed in endometrial cancer (EC) cell lines and at the mRNA level in primary patient tumours. Here, we show that p110ß protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear:cytoplasmic staining ratios were detected in high-grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] were measured in the nucleus of EC cells, and pharmacological and genetic approaches showed that its production was partly dependent upon p110ß activity. Using immunofluorescence staining, p110ß and PtdIns(3,4,5)P3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110ß inhibition led to a decrease in both 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110ß that may contribute to tumorigenesis in EC.This article has an associated First Person interview with Fatemeh Mazloumi Gavgani, joint first author of the paper.

Cholesterol-containing lipid nanodiscs promote an α-synuclein binding mode that accelerates oligomerization.

Dysregulation of the biosynthesis of cholesterol and other lipids has been implicated in many neurological diseases, including Parkinson's disease. Misfolding of α-synuclein (α-Syn), the main actor in Parkinson's disease, is associated with changes in a lipid environment. However, the exact molecular mechanisms underlying cholesterol effect on α-Syn binding to lipids as well as α-Syn oligomerization and fibrillation remain elusive, as does the relative importance of cholesterol compared to other factors. We probed the interactions and fibrillation behaviour of α-Syn using styrene-maleic acid nanodiscs, containing zwitterionic and anionic lipid model systems with and without cholesterol. Surface plasmon resonance and thioflavin T fluorescence assays were employed to monitor α-Syn binding, as well as fibrillation in the absence and presence of membrane models. 1 H-15 N-correlated NMR was used to monitor the fold of α-Syn in response to nanodisc binding, determining individual residue apparent affinities for the nanodisc-contained bilayers. The addition of cholesterol inhibited α-Syn interaction with lipid bilayers and, however, significantly promoted α-Syn fibrillation, with a more than a 20-fold reduction of lag times before fibrillation onset. When α-Syn bilayer interactions were analysed at an individual residue level by solution-state NMR, we observed two different effects of cholesterol. In nanodiscs made of DOPC, the addition of cholesterol modulated the NAC part of α-Syn, leading to stronger interaction of this region with the lipid bilayer. In contrast, in the nanodiscs comprising DOPC, DOPE and DOPG, the NAC part was mostly unaffected by the presence of cholesterol, while the binding of the N and the C termini was both inhibited.

SIX3 and SIX6 interact with GEMININ via C-terminal regions.

The histoarchitecture and function of eye and forebrain depend on a well-controlled balance between cell proliferation and differentiation. For example, the binding of the cell cycle regulator GEMININ to CDT1, which is a part of the pre-replication complex, promotes cell differentiation. Homeodomain transcription factors SIX3 and SIX6 also interact with GEMININ of which SIX3-GEMININ interaction promotes cell proliferation, whereas the nature of SIX6-GEMININ interaction has not been studied to date. We investigated SIX3/SIX6 and GEMININ interactions using bimolecular fluorescence complementation, surface plasmon resonance and isothermal titration calorimetry. Interactions between SIX3/SIX6 and GEMININ were detected in mammalian cells in culture. The presence of the C-terminal regions of SIX3 and SIX6 proteins, but not their SIX domains or homeodomains as previously thought, were required for interaction with GEMININ. Interestingly, the disordered C- and N- terminal regions of GEMININ were involved in binding to SIX3/SIX6. The coiled-coil region of GEMININ, which is the known protein-binding domain and also interacts with CDT1, was not involved in GEMININ-SIX3/SIX6 interaction. Using SPR and ITC, SIX3 bound GEMININ with a micromolar affinity and the binding stoichiometry was 1:2 (SIX3 - GEMININ). The present study gives new insights into the binding properties of SIX proteins, especially the role of their variable and disordered C-terminal regions.

A Case of Unilateral Hyperlucency of the Lung: a Rare Adult Occurrence of Swyer-James-MacLeod Syndrome.

Swyer-James-MacLeod syndrome is a rare pulmonary disorder that exhibits unilateral hyperlucency of a part of or the entire lung and because it is usually asimptomatic the diagnostic of this illness is discovered accidentally after a chest X-ray. Although the cause of this disorder is not completely understood, most authors believe that this is due to late sequela of infectious bronchiolitis that occurs during childhood. The symptoms presented by the individuals are often scarce at the moment of diagnostic, but a history of recurrent episodes of pulmonary infection accompanied by dyspnea, hemoptysis and/or chronic productive cough should raise the suspicion for such a diagnosis. Swyer-James-MacLeod syndrome can be interpreted as asthma or pulmonary embolism and this can result in inappropriate therapy. This case report describes a 38-year-old patient with chronic respiratory symptoms diagnosed on the findings of the chest X-ray and high resolution CT.

A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction.

Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1 in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110ß and its product PtdIns(3,4,5)P3 together with EBP1 in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdIns(3,4,5)P3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110ß and PtdIns(3,4,5)P3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1.