PHB Domain Research Articles

Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation

The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2TG) mice, PHB2S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.

In vivo characterization of a podocyte-expressed short podocin isoform

The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.

Preventive healthcare behavior: A hybrid systematic literature review (1998–2023)

AbstractPreventive healthcare behavior (PHB) refers to actions taken by consumers to avert possible incidences of lifestyle diseases. Over the years, diverse approaches have been used to comprehend the complex nature of PHB. This paper follows a three‐step process to examine work done in the PHB domain. At the outset, past literature was examined. This review included PHB models and frameworks, followed by a root‐cause analysis to identify factors that impacted PHB adoption. A systematic literature review (SLR) using a domain‐based hybrid review approach was the study's third and most crucial part. The SPAR‐4‐SLR (scientific procedures and rationales for systematic literature reviews) protocol was used to conduct the hybrid review, involving two separate review studies. In the first study, a bibliometric analysis was carried out, wherein a trend analysis was conducted on an initial pool of 1011 primary peer‐reviewed publications (1998–2023). The trend analysis was followed by a co‐citation network analysis of 39,608 secondary articles, which validated the importance of primary articles as indicated by the co‐citations in these secondary articles. Further, a purification process based on reliability, validity, and replicability criteria resulted in a final pool of 190 relevant articles. These articles were subjected to a thematic analysis. Next, a framework‐based review based on the theories, contexts, characteristics, and methods (TCCM) framework was conducted on the 190 articles. This step validated the primary study findings. Additionally, it examined and reported the underlying theories, context (country level), characteristics, and methods adopted by previous PHB studies. This analysis helps in indicating the future research agenda regarding PHB. Furthermore, the inferences drawn from the two studies were used to propose a conceptual framework for understanding consumers' PHB decisions based on the antecedents, decisions, and outcomes (ADO) framework. The framework posits that specific personal, demographic, cultural, social and socioeconomic factors are precursors to PHB adoption. This PHB adoption, in turn, has positive outcomes such as enhanced quality of life, consumer wellbeing, health promotion, health behavior change, and planning. The comprehensive review and proposed framework will significantly help advance knowledge about PHB. Apart from contributing to the academic literature, learnings from this study hold value for practitioners at the global level for designing actionable strategies for preventive healthcare products and services.

LINC00478-derived novel cytoplasmic lncRNA LacRNA stabilizes PHB2 and suppresses breast cancer metastasis via repressing MYC targets

BackgroundMetastasis is the predominant cause of mortality in patients with breast cancer. Long noncoding RNAs (lncRNAs) have been shown to drive important phenotypes in tumors, including invasion and metastasis. However, the lncRNAs involved in metastasis and their molecular and cellular mechanisms are still largely unknown.MethodsThe transcriptional and posttranscriptional processing of LINC00478-associated cytoplasmic RNA (LacRNA) was determined by RT-qPCR, semiquantitative PCR and 5′/3′ RACE. Paired-guide CRISPR/cas9 and CRISPR/dead-Cas9 systems was used to knock out or activate the expression of LacRNA. Cell migration and invasion assay was performed to confirm the phenotype of LacRNA. Tail vein model and mammary fat pad model were used for in vivo study. The LacRNA-PHB2-cMyc axis were screened and validated by RNA pulldown, mass spectrometry, RNA immunoprecipitation and RNA-seq assays.ResultsHere, we identified a novel cytoplasmic lncRNA, LacRNA (LINC00478-associated cytoplasmic RNA), derived from nucleus-located lncRNA LINC00478. The nascent transcript of LINC00478 full-length (LINC00478_FL) was cleaved and polyadenylated, simultaneously yielding 5′ ends stable expressing LacRNA, which is released into the cytoplasm, and long 3′ ends of nuclear-retained lncRNA. LINC00478_3′RNA was rapidly degraded. LacRNA significantly inhibited breast cancer invasion and metastasis in vitro and in vivo. Mechanistically, LacRNA physically interacted with the PHB domain of PHB2 through its 61–140-nt region. This specific binding affected the formation of the autophagy degradation complex of PHB2 and LC3, delaying the degradation of the PHB2 protein. Unexpectedly, LacRNA specifically interacted with PHB2, recruited c-Myc and promoted c-Myc ubiquitination and degradation. The negatively regulation of Myc signaling ultimately inhibited breast cancer metastasis. Furthermore, LacRNA and LacRNA-mediated c-Myc signaling downregulation are significantly associated with good clinical outcomes, take advantage of these factors we constructed a prognostic predict model.ConclusionTherefore, our findings propose LacRNA as a potential prognostic biomarker and a new therapeutic strategy.

Role of ERLINs in the Control of Cell Fate through Lipid Rafts.

ER lipid raft-associated protein 1 (ERLIN1) and 2 (ERLIN2) are 40 kDa transmembrane glycoproteins belonging to the family of prohibitins, containing a PHB domain. They are generally localized in the endoplasmic reticulum (ER), where ERLIN1 forms a heteroligomeric complex with its closely related ERLIN2. Well-defined functions of ERLINS are promotion of ER-associated protein degradation, mediation of inositol 1,4,5-trisphosphate (IP3) receptors, processing and regulation of lipid metabolism. Until now, ERLINs have been exclusively considered protein markers of ER lipid raft-like microdomains. However, under pathophysiological conditions, they have been described within mitochondria-associated endoplasmic reticulum membranes (MAMs), tethering sites between ER and mitochondria, characterized by the presence of specialized raft-like subdomains enriched in cholesterol and gangliosides, which play a key role in the membrane scrambling and function. In this context, it is emerging that ER lipid raft-like microdomains proteins, i.e., ERLINs, may drive mitochondria-ER crosstalk under both physiological and pathological conditions by association with MAMs, regulating the two main processes underlined, survival and death. In this review, we describe the role of ERLINs in determining cell fate by controlling the “interchange” between apoptosis and autophagy pathways, considering that their alteration has a significant impact on the pathogenesis of several human diseases.

Effect of Reprocessing Cycles on the Morphology and Mechanical Properties of a Poly(Propylene)/Poly(Hydroxybutyrate) Blend and its Nanocomposite

Abstract The use of polymer blends and composites based on fossil-based and bio-based polymers has become an important environmentally protective alternative for common use and disposable plastics applications such as packaging, bottles and trays. The disposal of these more degradable products, however, may also harm the environment and, therefore, recycling these systems becomes relevant. Recycling involves reprocessing which can significantly change the morphology and properties of polymeric products. Therefore, this study deals with the effects of reprocessing cycles on the properties and morphology of blends and nanocomposites based on fossil and bio-based polymers. The systems investigated were: a) neat polypropylene (PP), b) a polypropylene/poly(3-hydroxybutyrate) (PP/PHB) blend and c) PP/PHB/organoclay nanocomposite compatibilized with polypropylene-g-maleic anhydride (PP-g-MA) and erucamide. These materials were submitted to up to seven extrusion cycles in a single screw extruder operating at 60 rpm. Samples were taken after the first, third, fifth and seventh extrusion cycles and their tensile properties and morphology were determined. Scanning electron microscopy indicated that two phases were observed in the blend which showed spherical PHB domains. The addition of clay, PP-g-MA and erucamide improved the adhesion between the nanocomposites components. X-ray diffraction analysis showed that crystallinity tended to increase with the number of reprocessing cycles for all systems investigated up to the fifth cycle and then tended to decrease. A 10% crystallinity increase was observed for neat PP in the fifth cycle. In general, the tensile properties of all systems decreased with reprocessing and the highest losses were observed for the PP/PHB blend after seven processing cycles with 50% and 37% decreases in stress at break and elastic modulus, respectively. Impact strength of the PP matrix and of the PP/PHB blend tended to decrease with reprocessing, except for the nanocomposite which showed a slight increase especially after the seventh processing cycle in which an 18% increase in impact strength was observed.

Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2.

Bax inhibitor-1 (BI1) conveys anti-apoptotic signals for mitochondria while prohibitin 2 (PHB2) is implicated in sustaining mitochondrial morphology and function. However, their regulatory roles in acute kidney injury (AKI) are largely unknown.Methods: In human patients with AKI, levels of BI1 in urine and plasma were determined using ELISA. An experimental model of AKI was established using ATP depletion-mediated metabolic stress and ischemia-reperfusion injury (IRI) in primary tubule cells and BI1 transgenic mice, respectively. Western blots, ELISA, qPCR, immunofluorescence, RNA silencing, and domain deletion assay were employed to evaluate the roles of BI1 and PHB2 in the preservation of mitochondrial integrity.Results: Levels of BI1 in urine and plasma were decreased in patients with AKI and its expression correlated inversely with renal function. However, reconstitution of BI1 in a murine AKI model was capable of alleviating renal failure, inflammation and tubular death. Further molecular scrutiny revealed that BI1 preserved mitochondrial genetic integrity, reduced mitochondrial oxidative stress, promoted mitochondrial respiration, inhibited excessive mitochondrial fission, improved mitophagy and suppressed mitochondrial apoptosis. Intriguingly, levels of the mitochondria-localized PHB2 were sustained by BI1 and knockdown of PHB2 abolished the mitochondrial- and renal- protective properties of BI1. Furthermore, BI1 promoted PHB2 retention within mitochondria through direct interaction with cytoplasmic PHB2 to facilitate its mitochondrial import. This was confirmed by the observation that the C-terminus of BI1 and the PHB domain of PHB2 were required for the BI1-PHB2 cross-linking.Conclusion: Our data have unveiled an essential role of BI1 as a master regulator of renal tubule function through sustaining mitochondrial localization of PHB2, revealing novel therapeutic promises against AKI.

Manufacturing and Characterization of Toughened Poly(lactic acid) (PLA) Formulations by Ternary Blends with Biopolyesters.

Ternary blends with a constant poly(lactic acid) (PLA) content (60 wt %) and varying amounts of poly(3-hydroxybutyrate) (PHB) and poly(ε-caprolactone) (PCL) were manufactured by one step melt blending process followed by injection moulding, with the main aim of improving the low intrinsic toughness of PLA. Mechanical properties were obtained from tensile and Charpy impact tests. The miscibility and morphology of the system was studied by thermal analysis and field emission scanning electron microscopy (FESEM). The obtained results showed a clear phase separation, thus indicating poor miscibility between these three biopolyesters, i.e., PLA, the continuous component with dispersed PHB and PCL domains in the form of different sphere size. Nevertheless, the high fragility of PLA was remarkably reduced, as detected by the Charpy impact test. In accordance with the decrease in brittleness, a remarkable increase in elongation at break is achieved, with increasing PCL load due to its flexibility; in addition, increasing PCL load provides thermal stability at high temperatures. Thus, tailored materials can be manufactured by melt blending PLA, PHB, and PCL in different percentages to offer a wide range of biodegradable polymer blends.

Overcoming STC2 mediated drug resistance through drug and gene co-delivery by PHB-PDMAEMA cationic polyester in liver cancer cells

Stanniocalcin 2 (STC2) overexpression in hepatocellular carcinoma (HCC) could lead to poor prognosis, which might be due to its induced P-glycoprotein and Bcl-2 protein expression level increase. P-glycoprotein or membrane pump induced drug efflux and altered prosurvival Bcl-2 expression are key mechanisms for drug resistance leading to failure of chemotherapy in HCC. However, current strategy to overcome both P-glycoprotein and Bcl-2 protein induced drug resistance was rarely reported. In this work, we utilized an amphiphilic poly[(R)-3-hydroxybutyrate] (PHB)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) cationic polyester to encapsulate chemotherapeutic paclitaxel (PTX) in hydrophobic PHB domain and Bcl-2 convertor Nur77/ΔDBD gene (Nur77 without DNA binding domain for mitochondria localization) by formation of polyplex due to cationic PDMAEMA segment, to effectively inhibit the drug resistant HepG2/STC2 and SMCC7721/STC2 liver cancer cell growth. Thanks to the cationic nanoparticle complex formation ability and high transfection efficiency to express Bcl-2 conversion proteins, PHB-PDMAEMA/PTX@polyplex could partially impair P-glycoprotein induced PTX efflux and activate the apoptotic function of previous prosurvival Bcl-2 protein. This is the pioneer report of cationic amphiphilic polyester PHB-PDMAEMA to codeliver anticancer drug and therapeutic plasmid to overcome both pump and non-pump mediated chemotherapeutic resistance in liver cancer cells, which might be inspiring for the application of polyester in personalized cancer therapy.

MdHIR proteins repress anthocyanin accumulation by interacting with the MdJAZ2 protein to inhibit its degradation in apples

In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis.

Effects of Composition and Melting Time on the Phase Separation of Poly(3-hydroxybutyrate)/Poly(propylene carbonate) Blend Thin Films.

In this study, the effect of composition and melting time on the phase separation of poly(3-hydroxybutyrate)/poly(propylene carbonate) (PHB/PPC) blend thin films was investigated. Optical microscopy under phase contrast confirms the occurrence of phase separation of PHB/PPC blends at 190 °C. Polarized optical and scanning electron microscopies (POM and SEM) demonstrate that phase separation takes place along both horizontal and vertical film planes, which should be attributed to the two different interfaces and immiscible blends. A low PPC content (e.g. 30 wt %) results in the formation of compact PHB spherulites filling the whole space, whereas the noncrystallizable PPC spherical microdomains scatter in the PHB region, and their size shows a remarkable melting-time dependence. With the increasing PPC component and melting time, it is observed from POM that the separated PHB domains scattered in the continuous PPC phase, like the island structure. However, it can be revealed by SEM micrographs that the PHB thick domains are actually connected by its thin layer under the PPC layer. The real situation is, therefore, a large amount of PPC aggregates to the surface to form a network uplayer, whereas the PHB thick domains connected by its thin layer form a continuous PHB region, leading to a superimposed bilayer structure. There is also a small amount of PHB small domains scattered in the PHB phase. The PHB thick domains crystallize cooperatively with the PHB- or PHB-rich sublayer in a way just like the growth of pure PHB spherulites. This superimposed bilayer by interplay between phase separation and crystallization may provide availability to tailor the final structure and properties of crystalline/amorphous polymer blends.

Compatibilization of cellulose acetate/poly(3-hydroxybutyrate) blends by grafting copolymer

Blends of cellulose acetate (CA) and poly(3-hydroxybutyrate) (PHB) were prepared by the solvent-casting method in both the presence and the absence of a compatibilizer agent. The compatibilizer was synthesized by reactive processing between CA and PHB. From the differential scanning calorimetric analysis, it has been identified that CA/PHB blends are immiscible in the range of PHB content studied. The existence of interactions between the components was observed due to the displacement of glass transition temperature relative to PHB phase in the blend. The grafting copolymer promoted the increase of interactions between the phases specifically to compatibilized CA/40%wtPHB blend. This composition shows significant changes in crystalline percentage compared to the uncompatibilized blend. The existence of phase interactions modified the viscoelastic behavior of the compatibilized blends by introducing changes in the chain segment mobility due to the addition of compatibilizer as the dynamic mechanical analysis has pointed out. It was observed that different of the blends with lower PHB content, CA/40%wtPHB compatibilized blend with same compatibilizer content did not reach PHB saturation. This difference in behavior can be attributed to the fact that the content of compatibilizer agent used was the proper amount to promote both PHB domain size reduction and dispersion. POLYM. ENG. SCI., 2016. © 2016 Society of Plastics Engineers

Dissecting the molecular properties of prokaryotic flotillins.

Flotillins are universally conserved proteins that are present in all kingdoms of life. Recently it was demonstrated that the B. subtilis flotillin YuaG (FloT) has a direct influence on membrane domain formation by orchestrating lipid domains. Thereby it allocates a proper environment for diverse cellular machineries. YuaG creates platforms for signal transduction, processes crucial for biofilm formation, sporulation, competence, secretion, and others. Even though, flotillins are an emerging topic of research in the field of microbiology little is known about the molecular architecture of prokaryotic flotillins. All flotillins share common structural elements and are tethered to the membrane N’- terminally, followed by a so called PHB domain and a flotillin domain. We show here that prokaryotic flotillins are, similarly to eukaryotic flotillins, tethered to the membrane via a hairpin loop. Further it is demonstrated by sedimentation assays that B. subtilis flotillins do not bind to the membrane via their PHB domain contrary to eukaryotic flotillins. Size exclusion chromatography experiments, blue native PAGE and cross linking experiments revealed that B. subtilis YuaG can oligomerize into large clusters via the PHB domain. This illustrates an important difference in the setup of prokaryotic flotillins compared to the organization of eukaryotic flotillins.

Characterization of a short isoform of the kidney protein podocin in human kidney

BackgroundSteroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking.MethodsWe used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin.ResultsMass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform.ConclusionsA second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum.

ASURA (PHB2) Interacts with Scc1 through Chromatin

Sister chromatid cohesion mediated by the cohesin complex is essential for faithful chromosome segregation. Previously we reported that PHB2 (prohibitin2/ASURA), a multifunctional protein, has a role in sister chromatid cohesion. Nevertheless, how ASURA is involved in sister chromatid cohesion still remains unclear. The present co-immunoprecipitation analysis reveals that ASURA interacts with cohesin subunit Scc1 in vivo. We show that ASURA associates with chromatin in a similar manner as Scc1 throughout the cell cycle. Furthermore, our observation using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system indicates that ASURA is important for cohesin maintenance at early mitosis. We have also identified that the conserved PHB domain is responsible for chromatin targeting of ASURA. Our results suggest that the regulation of sister chromatid cohesion is mediated by ASURA binding to chromatin, where ASURA might be involved in cohesin protection through ASURA-Scc1 interactions.

Effects of Hydrogen Bond Intermolecular Interactions on the Crystal Spherulite of Poly(3-hydroxybutyrate) and Cellulose Acetate Butyrate Blends: Studied by FT-IR and FT-NIR Imaging Spectroscopy

The crystal melting behaviors of poly(3-hydroxybutyrate) (PHB) and cellulose acetate butyrate (CAB) blends were studied using infrared (IR) and near-infrared (NIR) imaging, which provided information about spherulite growth in dynamic blend systems. By analyzing the changes in the IR and NIR imaging spectra in the regions of the first and second overtones of the C═O stretching vibrations of PHB and CAB, the evolution of heterogeneous spherulite during the time-resolved isothermal crystallization process was explored. Time-resolved IR and NIR imaging and polarized microscopic studies detected the PHB domains are able to separate from the PHB/CAB blends early in the process. Principal component analysis (PCA) was used to classify the distribution of the different morphologies of spherulite. The first principal component suggests that the discrimination of the imaging spectra relies largely upon the crystallinity, while the second principal component indicates the variations in the amorphous portion of PHB, ...

Gene expression profiles of prohibitin in testes of Octopus tankahkeei (ot-phb) revealing its possible role during spermiogenesis

Prohibitin is essential for intracellular homeostasis and stabilization of mitochondrial respiratory chain complexes. To explore its functions during spermiogenesis of Octopus tankahkeei (O. tankahkeei), we have cloned and sequenced the cDNA of this mammalian PHB homologue (termed ot-PHB) from the testes of O. tankahkeei. The 1165bp ot-phb cDNA contains a 100bp 5' UTR, a 882bp open reading frame and a 183bp 3' UTR. The putative ot-PHB protein owns a transmembrane domain from 6 to 31 amino acid (aa) and a putative PHB domain from 26 to 178 aa. Protein alignment demonstrated that ot-PHB had 73.3, 73.6, 74.0, 75.1, and 45.4% identity with its homologues in Homo sapiens, Mus muculus, Danio rerio, Xenopus tropicalis and Trypanosoma brucei, respectively. Tissue distribution profile analysis revealed its presence in all the tissues examined. In situ hybridization in spermiogenic cells demonstrated that ot-phb was expressed moderately at the beginning of the spermiogenesis. The abundance of transcripts increased in intermediate spermatids and in drastically remodeling final spermatids. In mature spermatozoa, the residuary transcripts concentrated around the chondriosomal mantle where mitochondria assemble around. In summary, the expression of ot-phb during spermiogenesis implicates a potential function of this protein during mitochondrial ubiquitination. It is the first time to implicate the role of prohibitin in cephalopod spermiogenesis.

In Situ Orientation Studies of a Poly(3-hydroxybutyrate)/Poly(ε-caprolactone) Blend by Rheo-Optical Fourier Transform Infrared Spectroscopy and Two-Dimensional Correlation Spectroscopic Analysis

In the present study, the orientation of a poly(3-hydroxybutyrate) (PHB)/poly(epsilon-caprolactone) (PCL) blend was monitored during uniaxial elongation by rheo-optical Fourier transform infrared (FT-IR) spectroscopy and analyzed by generalized two-dimensional correlation spectroscopy (2D-COS). The dichroism of the delta(CH(2)) absorption bands of PHB and PCL was employed to determine the polymer chain orientation in the PHB/PCL blend during the elongation up to 267% strain. From the PHB and PCL specific orientation functions it was derived that the PCL chains orient into the drawing direction while the PHB chains orient predominantly perpendicular to the applied strain. To extract more detailed information about the polymer orientation during uniaxial elongation, 2D-COS analysis was employed for the dichroic difference of the polarization spectra recorded during the drawing process. In the corresponding synchronous and asynchronous 2D correlation plots, absorption bands characteristic of the crystalline and amorphous regions of PHB and PCL were separated. Furthermore, the 2D-COS analysis revealed that during the mechanical treatment the PCL domains orient before the PHB domains.

Lipid–Protein Interactions along the Slit Diaphragm of Podocytes

Podocytes are visceral epithelial cells supporting the function of the glomerular filter. Interdigitating foot processes of podocytes enwrap the glomerular capillaries and are connected by a highly specialized cell junction, the slit diaphragm. Signal transduction at the slit diaphragm is essential for the proper function of the kidney filtration barrier. The slit diaphragm constitutes a dynamic multiprotein signaling complex that contains structural proteins, receptors, signaling adaptors, ion channels, and scaffolding proteins. Function of some of these proteins requires cholesterol attached to the multiprotein complex. Recruitment of cholesterol is achieved through the PHB domain protein podocin, a member of a novel family of lipid-binding proteins that are conserved through evolution. The finding that cholesterol interaction regulates the activity of ion channels at the glomerular filtration barrier has important implications for renal physiology and pathophysiology.

Competition between targeting signals in hybrid proteins provides information on their relative in vivo affinities for subcellular compartments

After their translation and folding in the cytoplasm, proteins may be imported into an organelle, associate with a membrane, or rather become part of large, highly localised cytoplasmic structures such as the cytoskeleton. The localisation of a protein is governed by the strength of binding to its immediate target, such as an import receptor for an organelle or a major component of the cytoskeleton, e.g. actin. We have experimentally provided a set of actin-binding proteins with competing targeting information and expressed them at various concentrations to analyse the strength of the signal that governs their subcellular localisation. Our microscopic observations indicate that organellar sorting signals override the targeting preference of most cytoskeletal proteins. Among these signals, the nuclear localisation signal of SV40 is strongest, followed by the oligomerised PHB domain that targets vacuolin to the endosomal surface, and finally the tripeptide SKL mediating transport into the peroxisome. The actin-associated protein coronin, however, can only be misled by the nuclear localisation signal. Interestingly, the targeting behaviour of this model set of hybrid proteins in living Dictyostelium amoebae correlates surprisingly well with the affinities of their constituent signals derived from in vitro experiments conducted in various other organisms. Accordingly, this approach allows estimating the in vivo affinity of a protein to its target even if the latter is not known, as in the case of vacuolin.