Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation.

Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.

Characterization of Anorexia Nervosa on Social Media: Textual, Visual, Relational, Behavioral, and Demographical Analysis.

BACKGROUND: Eating disorders are psychological conditions characterized by unhealthy eating habits. Anorexia nervosa (AN) is defined as the belief of being overweight despite being dangerously underweight. The psychological signs involve emotional and behavioral issues. There is evidence that signs and symptoms can manifest on social media, wherein both harmful and beneficial content is shared daily. OBJECTIVE: This study aims to characterize Spanish-speaking users showing anorexia signs on Twitter through the extraction and inference of behavioral, demographical, relational, and multimodal data. By using the transtheoretical model of health behavior change, we focus on characterizing and comparing users at the different stages of the model for overcoming AN, including treatment and full recovery periods. METHODS: We analyzed the writings, posting patterns, social relationships, and images shared by Twitter users who underwent different stages of anorexia nervosa and compared the differences among users going through each stage of the illness and users in the control group (ie, users without AN). We also analyzed the topics of interest of their followees (ie, users followed by study participants). We used a clustering approach to distinguish users at an early phase of the illness (precontemplation) from those that recognize that their behavior is problematic (contemplation) and generated models for the detection of tweets and images related to AN. We considered two types of control users-focused control users, which are those that use terms related to anorexia, and random control users. RESULTS: We found significant differences between users at each stage of the recovery process (P<.001) and control groups. Users with AN tweeted more frequently at night, with a median sleep time tweets ratio (STTR) of 0.05, than random control users (STTR=0.04) and focused control users (STTR=0.03). Pictures were relevant for the characterization of users. Focused and random control users were characterized by the use of text in their profile pictures. We also found a strong polarization between focused control users and users in the first stages of the disorder. There was a strong correlation among the shared interests between users with AN and their followees (ρ=0.96). In addition, the interests of recovered users and users in treatment were more highly correlated to those corresponding to the focused control group (ρ=0.87 for both) than those of AN users (ρ=0.67), suggesting a shift in users' interest during the recovery process. CONCLUSIONS: We mapped the signs of AN to social media context. These results support the findings of previous studies that focused on other languages and involved a deep analysis of the topics of interest of users at each phase of the disorder. The features and patterns identified provide a basis for the development of detection tools and recommender systems.

Correction: Characterization of Anorexia Nervosa on Social Media: Textual, Visual, Relational, Behavioral, and Demographical Analysis.

[This corrects the article DOI: 10.2196/25925.].

Detection of Suicidal Ideation on Social Media: Multimodal, Relational, and Behavioral Analysis.

BACKGROUND: Suicide risk assessment usually involves an interaction between doctors and patients. However, a significant number of people with mental disorders receive no treatment for their condition due to the limited access to mental health care facilities; the reduced availability of clinicians; the lack of awareness; and stigma, neglect, and discrimination surrounding mental disorders. In contrast, internet access and social media usage have increased significantly, providing experts and patients with a means of communication that may contribute to the development of methods to detect mental health issues among social media users. OBJECTIVE: This paper aimed to describe an approach for the suicide risk assessment of Spanish-speaking users on social media. We aimed to explore behavioral, relational, and multimodal data extracted from multiple social platforms and develop machine learning models to detect users at risk. METHODS: We characterized users based on their writings, posting patterns, relations with other users, and images posted. We also evaluated statistical and deep learning approaches to handle multimodal data for the detection of users with signs of suicidal ideation (suicidal ideation risk group). Our methods were evaluated over a dataset of 252 users annotated by clinicians. To evaluate the performance of our models, we distinguished 2 control groups: users who make use of suicide-related vocabulary (focused control group) and generic random users (generic control group). RESULTS: We identified significant statistical differences between the textual and behavioral attributes of each of the control groups compared with the suicidal ideation risk group. At a 95% CI, when comparing the suicidal ideation risk group and the focused control group, the number of friends (P=.04) and median tweet length (P=.04) were significantly different. The median number of friends for a focused control user (median 578.5) was higher than that for a user at risk (median 372.0). Similarly, the median tweet length was higher for focused control users, with 16 words against 13 words of suicidal ideation risk users. Our findings also show that the combination of textual, visual, relational, and behavioral data outperforms the accuracy of using each modality separately. We defined text-based baseline models based on bag of words and word embeddings, which were outperformed by our models, obtaining an increase in accuracy of up to 8% when distinguishing users at risk from both types of control users. CONCLUSIONS: The types of attributes analyzed are significant for detecting users at risk, and their combination outperforms the results provided by generic, exclusively text-based baseline models. After evaluating the contribution of image-based predictive models, we believe that our results can be improved by enhancing the models based on textual and relational features. These methods can be extended and applied to different use cases related to other mental disorders.

Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation.

Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

The Hydrophilic C-terminus of Yeast Plasma-membrane Na+/H+ Antiporters Impacts Their Ability to Transport K.

Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

Human EWS-FLI protein levels and neomorphic functions show a complex, function-specific dose-response relationship in Drosophila.

Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing's sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI's high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS's lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAµSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAµSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAµSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI 'high' and 'low' states that have been reported and are suspected to be important for EwS in humans.

The toxic effects of yeast Ppz1 phosphatase are counteracted by subcellular relocalization mediated by its regulatory subunit Hal3.

Overexpression of Saccharomyces cerevisiae protein phosphatase Ppz1 strongly impairs cell growth. Ppz1 is negatively regulated by its subunit Hal3, and Hal3 overexpression fully counteracts the toxic effects derived from high levels of the phosphatase. We show that Ppz1 localizes at the plasma membrane, and that co-expression of Hal3 recruits Ppz1 to internal membranes (mostly vacuolar). This effect is not observed in a catalytically impaired mutant of Ppz1. Disruption of intracellular trafficking by deletion of the ESCRT-0 component VPS27 abolishes both Hal3-mediated relocalization of Ppz1 and normalization of cell growth, without affecting Ppz1 protein levels. We propose that Hal3 counteracts the toxic effects caused by excess of Ppz1 not only by inhibiting its enzymatic activity but also by recruiting the phosphatase to internal structures.

Functional mapping of the N-terminal region of the yeast moonlighting protein Sis2/Hal3 reveals crucial residues for Ppz1 regulation.

The function of the Saccharomyces cerevisiae Ppz1 phosphatase is controlled by its inhibitory subunit Hal3. Hal3 is a moonlighting protein, which associates with Cab3 to form a decarboxylase involved in the CoA biosynthetic pathway. Hal3 is composed by a conserved core PD region, required for both Ppz1 regulation and CoA biosynthesis, a long N-terminal extension, and an acidic C-terminal tail. Cab3 has a similar structure, but it is not a Ppz1 inhibitor. We show here that deletion or specific mutations in a short region of the N-terminal extension of Hal3 compromise its function as a Ppz1 inhibitor in vivo and in vitro without negatively affecting its ability to interact with the phosphatase. This study defines a R-K-X(3) -VTFS- sequence whose presence explains the unexpected ability of Cab3 (but not Hal3) to regulate Ppz1 function in Candida albicans. This sequence is conserved in a subset of fungi and it could serve to estimate the relevance of Hal3 or Cab3 proteins in regulating fungal Ppz enzymes. We also show that the removal of the motif moderately affects both Ppz1 intracellular relocalization and counteraction of toxicity in cells overexpressing the phosphatase. Thus, our work contributes to our understanding of the regulation of Ppz phosphatases, which are determinants for virulence in some pathogenic fungi.

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+ /H+ antiporter NHA2.

The human Na+ /H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity.

The Toxic Effects of Ppz1 Overexpression Involve Nha1-Mediated Deregulation of K+ and H+ Homeostasis.

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation.

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.

Physicochemical, thermal and mechanical characterization study of perimysial collagen of two bovine muscles.

Chemical, thermal and mechanical collagen characteristics of intramuscular perimysial connective tissue (IMCT) from bovine Semitendinosus (ST) and Pectoralis profundus (PP) muscles were studied. Furthermore, these collagen characteristics in presence/absence of other extracellular matrix components were analyzed for both muscles. Differences between muscles were observed for collagen content; all IMCT-PP perimysial samples were higher than ST samples. In addition, for both muscles, IMCT-alkali resistant samples allowed the highest trypsin soluble collagen. The main differences between muscles were recorder for thermal and mechanical properties. The denaturation of collagen in the perimysium evidenced differences in total denaturation energy (ΔH) and peak temperatures (Tp). The ΔH resulted higher for IMCT-PP than for IMCT-ST tissues in all samples. By the tensile test it was observed that the maximum loads were constant and higher in all PP samples. In the FTIR assay, the peaks for the main amides were registered in both tissues. However, slight differences between ST and PP-IMCT were detected on hydrogen bond interactions and in secondary structure of the protein. The results reinforce the hypothesis of the presence of different IMCT-perimysial-collagen pools. In this study, chemical, thermal and mechanical characteristics were considered and quantified. However, the mechanical function and development of muscle in-vivo could be the main influence on the extracellular collagen characteristics as well as its interactions with non-collagen compounds. Its formation is essential for muscle function.

Ser/Thr protein phosphatases in fungi: structure, regulation and function.

Reversible phospho-dephosphorylation of proteins is a major mechanism for the control of cellular functions. By large, Ser and Thr are the most frequently residues phosphorylated in eukar-yotes. Removal of phosphate from these amino acids is catalyzed by a large family of well-conserved enzymes, collectively called Ser/Thr protein phosphatases. The activity of these enzymes has an enormous impact on cellular functioning. In this work we pre-sent the members of this family in S. cerevisiae and other fungal species, and review the most recent findings concerning their regu-lation and the roles they play in the most diverse aspects of cell biology.

Specific effects on strength and heat stability of intramuscular connective tissue during long time low temperature cooking.

Long-time low-temperature (LTLT) cooking of meat is known to produce a tender product. The current work tested the hypothesis that LTLT cooking for periods of up to 24 h at 60 °C reduces the contribution of intramuscular connective tissue to cooked meat toughness. Tensile tests on perimysium excised after cooking showed that its strength diminished with cooking time, although not as markedly as the Warner-Bratzler peak force measure of toughness. A gradually increasing susceptibility to trypsin digestion with increasing heating time demonstrated that there was a slow and gradual increase in the proportion of denatured collagen in the perimysium. Differential scanning calorimetry on perimysium excised after cooking showed an endothermic peak representing the denaturation of the collagen not already denatured on cooking. With increasing cooking time, the energy per milligram of collagen necessary to denature this remaining fraction increased. These results support the hypothesis that there is both an easily destabilized and more resistant fractions of the collagen in intramuscular connective tissue.

In vitro and in vivo evaluation of a dextran-graft-polybutylmethacrylate copolymer coated on CoCr metallic stent.

Introduction: The major complications of stent implantation are restenosis and late stent thrombosis. PBMA polymers are used for stent coating because of their mechanical properties. We previously synthesized and characterized Dextrangraft-polybutylmethacrylate copolymer (Dex-PBMA) as a potential stent coating. In this study, we evaluated the haemocompatibility and biocompatibility properties of Dex-PBMA in vitro and in vivo. Methods: Here, we investigated: (1) the effectiveness of polymer coating under physiological conditions and its ability to release Tacrolimus®, (2) the capacity of Dex-PBMA to inhibit Staphylococcus aureus adhesion, (3) the thrombin generation and the human platelet adhesion in static and dynamic conditions, (4) the biocompatibility properties in vitro on human endothelial colony forming cells ( ECFC) and on mesenchymal stem cells (MSC) and in vivo in rat models, and (5) we implanted Dex-PBMA and Dex-PBMATAC coated stents in neointimal hyperplasia restenosis rabbit model. Results: Dex-PBMA coating efficiently prevented bacterial adhesion and release Tacrolimus®. Dex-PBMA exhibit haemocompatibility properties under flow and ECFC and MSC compatibility. In vivo, no pathological foreign body reaction was observed neither after intramuscular nor intravascular aortic implantation. After Dex-PBMA and Dex-PBMATAC coated stents 30 days implantation in a restenosis rabbit model, an endothelial cell coverage was observed and the lumen patency was preserved. Conclusion: Based on our findings, Dex-PBMA exhibited vascular compatibility and can potentially be used as a coating for metallic coronary stents.

The thermal shrinkage force in perimysium from different beef muscles is not affected by post-mortem ageing.

Differences in the thermal shrinkage and collagen solubility between bovine Semitendinosus (ST) and Pectoralis profundus (PP) muscles and their interactions with ageing were evaluated by studying collagen solubility, hydrothermal isometric tension and thermal denaturation properties of intramuscular connective tissue after 5-20days post-mortem storage at 4°C. Collagen solubility was higher in ST than in PP muscle at 5-13days, but the differences between the two muscles decreased at longer ageing times. A small decrease in the peak denaturation temperature of perimysium occurred with increasing ageing times in both muscles. Maximum force in isometrically-heated perimysium was broadly equivalent in both muscles. Although the amount and solubility of collagen varies between muscles and ageing decreases the stability of some of the collagen, thermal shrinkage forces in heated perimysium are not significantly diminished by ageing. These findings support the idea of one collagen fraction easily degraded by ageing and heat, and another more resistant fraction that determines the physical properties of the tissue after ageing and cooking.

Mutations at the hydrophobic core affect Hal3 trimer stability, reducing its Ppz1 inhibitory capacity but not its PPCDC moonlighting function.

S. cerevisiae Hal3 (ScHal3) is a moonlighting protein that, is in its monomeric state, regulates the Ser/Thr protein phosphatase Ppz1, but also joins ScCab3 (and in some instances the Hal3 paralog Vhs3) to form an unusual heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. PPCDC is required for CoA biosynthesis and in most eukaryotes is a homotrimeric complex with three identical catalytic sites at the trimer interfaces. However, in S. cerevisiae the heterotrimeric arrangement results in a single functional catalytic center. Importantly, the specific structural determinants that direct Hal3's oligomeric state and those required for Ppz1 inhibition remain largely unknown. We mutagenized residues in the predicted hydrophobic core of ScHal3 (L403-L405) and the plant Arabidopsis thaliana Hal3 (AtHal3, G115-L117) oligomers and characterized their properties as PPCDC components and, for ScHal3, also as Ppz1 inhibitor. We found that in AtHal3 these changes do not affect trimerization or PPCDC function. Similarly, mutation of ScHal3 L403 has no effect. In contrast, ScHal3 L405E fails to form homotrimers, but retains the capacity to bind Cab3-explaining its ability to rescue a hal3 vhs3 synthetically lethal mutation. Remarkably, the L405E mutation decreases Hal3's ability to interact with and to inhibit Ppz1, confirming the importance of the oligomer/monomer equilibrium in Hal3's Ppz1 regulating function.

Differences in the energetics of collagen denaturation in connective tissue from two muscles.

The thermal denaturation of collagen the perimysium of intramuscular connective tissue isolated from bovine Semitendinosus (ST) and Perctoralis profundus (PP) muscles was investigated using a range of heating rates in differential scanning calorimetry (DSC) and analyzed by application of the Kissinger-Akahira-Sunose (KAS) and Lumry-Eyring models. Thermograms showed a broadening of endotherms and a shift towards higher temperatures as the thermal scanning rate increased. These features are consistent with the two-step process of a reversible transition between native and unfolded collagen molecules followed by an irreversible transition between unfolded and denatured states. There were small differences between muscles in the onset temperatures of the thermal transitions at heating rates of 0.5 K min-1, while both the KAS and Lumry-Eyring models yielded similar values for the effective activation energy of the whole two-step process, the Lumry-Eyring model allowed a greater insight into differences in the reversible and irreversible steps between the perimysium both muscles. Reversible unwinding of the triple-helical collagen molecules in the perimysium from ST muscle required more energy than in PP muscle. It is speculated that the presence of large amounts of elastin in the perimysium of ST muscles may influence this due to a protein crowding mechanism, or by affecting the covalent cross-linking of the collagen.