Polarization of the endoplasmic reticulum by ER-septin tethering.

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Protein Aggregation Triggered by Rewired Protein Homeostasis during Neuronal Differentiation.

In this issue of Molecular Cell, Vonk et al. (2020) and Thiruvalluvan et al. (2020) identify key chaperones that confer resistance to protein aggregation in neural stem cells and become reduced upon differentiation.

Shift of the insoluble content of the proteome in the aging mouse brain.

During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as Caenorhabditis elegans, proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals. To uncover age-dependent changes in the insoluble portion of a mammalian proteome, we analyzed the detergent-insoluble fraction of mouse brain tissue by mass spectrometry. We identified a group of 171 proteins, including the small heat shock protein α-crystallin, that become enriched in the detergent-insoluble fraction obtained from old mice. To enhance our ability to detect features associated with proteins in that fraction, we complemented our data with a meta-analysis of studies reporting the detergent-insoluble proteins in various mouse models of aging and neurodegeneration. Strikingly, insoluble proteins from young and old mice are distinct in several features in our study and across the collected literature data. In younger mice, proteins are more likely to be disordered, part of membraneless organelles, and involved in RNA binding. These traits become less prominent with age, as an increased number of structured proteins enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a group of proteins with specific features to become detergent-insoluble. Importantly, these features are not consistent with those associated with proteins driving membraneless organelle formation. We see no evidence in our system of a general increase of condensate proteins in the detergent-insoluble fraction with age.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

GraPES: The Granule Protein Enrichment Server for prediction of biological condensate constituents.

Phase separation-based condensate formation is a novel working paradigm in biology, helping to rationalize many important cellular phenomena including the assembly of membraneless organelles. Uncovering the functional impact of cellular condensates requires a better knowledge of these condensates' constituents. Herein, we introduce the webserver GraPES (Granule Protein Enrichment Server), a user-friendly online interface containing the MaGS and MaGSeq predictors, which provide propensity scores for proteins' localization into cellular condensates. Our webpage contains models trained on human (Homo sapiens) and yeast (Saccharomyces cerevisiae) stress granule proteins. MaGS utilizes experimentally-based protein features for prediction, whereas MaGSeq is an entirely protein sequence-based implementation. GraPES is implemented in HTML/CSS and Javascript and is freely available for public use at https://grapes.msl.ubc.ca/. Documentation for using the provided webtools, descriptions of their methodology, and implementation notes can be found on the webpage.

Protein interaction networks in neurodegenerative diseases: From physiological function to aggregation.

The accumulation of protein inclusions is linked to many neurodegenerative diseases that typically develop in older individuals, due to a combination of genetic and environmental factors. In rare familial neurodegenerative disorders, genes encoding for aggregation-prone proteins are often mutated. While the underlying mechanism leading to these diseases still remains to be fully elucidated, efforts in the past 20 years revealed a vast network of protein-protein interactions that play a major role in regulating the aggregation of key proteins associated with neurodegeneration. Misfolded proteins that can oligomerize and form insoluble aggregates associate with molecular chaperones and other elements of the proteolytic machineries that are the frontline workers attempting to protect the cells by promoting clearance and preventing aggregation. Proteins that are normally bound to aggregation-prone proteins can become sequestered and mislocalized in protein inclusions, leading to their loss of function. In contrast, mutations, posttranslational modifications, or misfolding of aggregation-prone proteins can lead to gain of function by inducing novel or altered protein interactions, which in turn can impact numerous essential cellular processes and organelles, such as vesicle trafficking and the mitochondria. This review examines our current knowledge of protein-protein interactions involving several key aggregation-prone proteins that are associated with Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. We aim to provide an overview of the protein interaction networks that play a central role in driving or mitigating inclusion formation, while highlighting some of the key proteomic studies that helped to uncover the extent of these networks.

Proteomic analysis reveals the direct recruitment of intrinsically disordered regions to stress granules in S. cerevisiae.

Stress granules (SGs) are stress-induced membraneless condensates that store non-translating mRNA and stalled translation initiation complexes. Although metazoan SGs are dynamic compartments where proteins can rapidly exchange with their surroundings, yeast SGs seem largely static. To gain a better understanding of yeast SGs, we identified proteins that sediment after heat shock using mass spectrometry. Proteins that sediment upon heat shock are biased toward a subset of abundant proteins that are significantly enriched in intrinsically disordered regions (IDRs). Heat-induced SG localization of over 80 proteins were confirmed using microscopy, including 32 proteins not previously known to localize to SGs. We found that several IDRs were sufficient to mediate SG recruitment. Moreover, the dynamic exchange of IDRs can be observed using fluorescence recovery after photobleaching, whereas other components remain immobile. Lastly, we showed that the IDR of the Ubp3 deubiquitinase was critical for yeast SG formation. This work shows that IDRs can be sufficient for SG incorporation, can remain dynamic in vitrified SGs, and can play an important role in cellular compartmentalization upon stress.This article has an associated First Person interview with the first author of the paper.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Unfolding or aggregation, that is the question.

Cellular processes accompanying protein aggregation are diverse and entangled, making it difficult to investigate the underlying molecular processes in a time-resolved way. Gottlieb, Thompson, and colleagues address this shortcoming using a chemical biology approach to monitor ubiquitination within the first 10 min after the initiation of protein aggregation. Intriguingly, unfolding rather than aggregation seems to trigger the observed events. This work might provide a method to answer open questions regarding the regulation of the proteostasis network upon protein misfolding.

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis (i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1 In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues.

Insulin-like growth factor 1 receptor stabilizes the ETV6-NTRK3 chimeric oncoprotein by blocking its KPC1/Rnf123-mediated proteasomal degradation.

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

The diversity of ubiquitin recognition: hot spots and varied specificity.

Ubiquitin is attached to a large number of proteins and gives rise to signaling events that modulate many cellular functions. These signals are often based on the recognition of polyubiquitin chains, which are produced in a variety of lengths and linkage patterns. In addition, proteins that are similar to ubiquitin in structure and function are often recognized by an overlapping set of partners. Research over the past several years has expanded our understanding of how ubiquitin and ubiquitin-like proteins are recognized. Most interactions occur at a few distinct surface areas; however, individual binding partners have specific, unique contacts that impart specificity. In this review, we summarize available information to facilitate comparisons across the ubiquitin-like family.

A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition - one to remove cohesin and the second to target Ty1-IN to chromatin.

Mapping the Broad Structural and Mechanical Properties of Amyloid Fibrils.

Amyloids are fibrillar nanostructures of proteins that are assembled in several physiological processes in human cells (e.g., hormone storage) but also during the course of infectious (prion) and noninfectious (nonprion) diseases such as Creutzfeldt-Jakob and Alzheimer's diseases, respectively. How the amyloid state, a state accessible to all proteins and peptides, can be exploited for functional purposes but also have detrimental effects remains to be determined. Here, we measure the nanomechanical properties of different amyloids and link them to features found in their structure models. Specifically, we use shape fluctuation analysis and sonication-induced scission in combination with full-atom molecular dynamics simulations to reveal that the amyloid fibrils of the mammalian prion protein PrP are mechanically unstable, most likely due to a very low hydrogen bond density in the fibril structure. Interestingly, amyloid fibrils formed by HET-s, a fungal protein that can confer functional prion behavior, have a much higher Young's modulus and tensile strength than those of PrP, i.e., they are much stiffer and stronger due to a tighter packing in the fibril structure. By contrast, amyloids of the proteins RIP1/RIP3 that have been shown to be of functional use in human cells are significantly stiffer than PrP fibrils but have comparable tensile strength. Our study demonstrates that amyloids are biomaterials with a broad range of nanomechanical properties, and we provide further support for the strong link between nanomechanics and ß-sheet characteristics in the amyloid core.

Ty1 Integrase Interacts with RNA Polymerase III-specific Subcomplexes to Promote Insertion of Ty1 Elements Upstream of Polymerase (Pol) III-transcribed Genes.

Retrotransposons are eukaryotic mobile genetic elements that transpose by reverse transcription of an RNA intermediate and are derived from retroviruses. The Ty1 retrotransposon of Saccharomyces cerevisiae belongs to the Ty1/Copia superfamily, which is present in every eukaryotic genome. Insertion of Ty1 elements into the S. cerevisiae genome, which occurs upstream of genes transcribed by RNA Pol III, requires the Ty1 element-encoded integrase (IN) protein. Here, we report that Ty1-IN interacts in vivo and in vitro with RNA Pol III-specific subunits to mediate insertion of Ty1 elements upstream of Pol III-transcribed genes. Purification of Ty1-IN from yeast cells followed by mass spectrometry (MS) analysis identified an enrichment of peptides corresponding to the Rpc82/34/31 and Rpc53/37 Pol III-specific subcomplexes. GFP-Trap purification of multiple GFP-tagged RNA Pol III subunits from yeast extracts revealed that the majority of Pol III subunits co-purify with Ty1-IN but not two other complexes required for Pol III transcription, transcription initiation factors (TF) IIIB and IIIC. In vitro binding studies with bacterially purified RNA Pol III proteins demonstrate that Rpc31, Rpc34, and Rpc53 interact directly with Ty1-IN. Deletion of the N-terminal 280 amino acids of Rpc53 abrogates insertion of Ty1 elements upstream of the hot spot SUF16 tRNA locus and abolishes the interaction of Ty1-IN with Rpc37. The Rpc53/37 complex therefore has an important role in targeting Ty1-IN to insert Ty1 elements upstream of Pol III-transcribed genes.

Cardiac Ryanodine Receptor (Ryr2)-mediated Calcium Signals Specifically Promote Glucose Oxidation via Pyruvate Dehydrogenase.

Cardiac ryanodine receptor (Ryr2) Ca2+ release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause or consequence of heart failure. Previous in vitro studies revealed that Ca2+ flux into the mitochondria helps pace oxidative metabolism, but there is limited in vivo evidence supporting this concept. Here, we studied heart-specific, inducible Ryr2 haploinsufficient (cRyr2Δ50) mice with a stable 50% reduction in Ryr2 protein. This manipulation decreased the amplitude and frequency of cytosolic and mitochondrial Ca2+ signals in isolated cardiomyocytes, without changes in cardiomyocyte contraction. Remarkably, in the context of well preserved contractile function in perfused hearts, we observed decreased glucose oxidation, but not fat oxidation, with increased glycolysis. cRyr2Δ50 hearts exhibited hyperphosphorylation and inhibition of pyruvate dehydrogenase, the key Ca2+-sensitive gatekeeper to glucose oxidation. Metabolomic, proteomic, and transcriptomic analyses revealed additional functional networks associated with altered metabolism in this model. These results demonstrate that Ryr2 controls mitochondrial Ca2+ dynamics and plays a specific, critical role in promoting glucose oxidation in cardiomyocytes. Our findings indicate that partial RYR2 loss is sufficient to cause metabolic abnormalities seen in heart disease.

Tuning the proteasome to brighten the end of the journey.

Degradation by the proteasome is the fate for a large portion of cellular proteins, and it plays a major role in maintaining protein homeostasis, as well as in regulating many cellular processes like cell cycle progression. A decrease in proteasome activity has been linked to aging and several age-related neurodegenerative pathologies and highlights the importance of the ubiquitin proteasome system regulation. While the proteasome has been traditionally viewed as a constitutive element of proteolysis, major studies have highlighted how different regulatory mechanisms can impact its activity. Importantly, alterations of proteasomal activity may have major impacts for its function and in therapeutics. On one hand, increasing proteasome activity could be beneficial to prevent the age-related downfall of protein homeostasis, whereas inhibiting or reducing its activity can prevent the proliferation of cancer cells.

System-wide analysis reveals intrinsically disordered proteins are prone to ubiquitylation after misfolding stress.

Damaged and misfolded proteins that are no longer functional in the cell need to be eliminated. Failure to do so might lead to their accumulation and aggregation, a hallmark of many neurodegenerative diseases. Protein quality control pathways play a major role in the degradation of these proteins, which is mediated mainly by the ubiquitin proteasome system. Despite significant focus on identifying ubiquitin ligases involved in these pathways, along with their substrates, a systems-level understanding of these pathways has been lacking. For instance, as misfolded proteins are rapidly ubiquitylated, unconjugated ubiquitin is rapidly depleted from the cell upon misfolding stress; yet it is unknown whether certain targets compete more efficiently to be ubiquitylated. Using a system-wide approach, we applied statistical and computational methods to identify characteristics enriched among proteins that are further ubiquitylated after heat shock. We discovered that distinct populations of structured and, surprisingly, intrinsically disordered proteins are prone to ubiquitylation. Proteomic analysis revealed that abundant and highly structured proteins constitute the bulk of proteins in the low-solubility fraction after heat shock, but only a portion is ubiquitylated. In contrast, ubiquitylated, intrinsically disordered proteins are enriched in the low-solubility fraction after heat shock. These proteins have a very low abundance in the cell, are rarely encoded by essential genes, and are enriched in binding motifs. In additional experiments, we confirmed that several of the identified intrinsically disordered proteins were ubiquitylated after heat shock and demonstrated for two of them that their disordered regions are important for ubiquitylation after heat shock. We propose that intrinsically disordered regions may be recognized by the protein quality control machinery and thereby facilitate the ubiquitylation of proteins after heat shock.

Proteomics propels protein degradation studies in San Diego.

Exquisite in vitro biochemical examinations of protein ubiquitylation and degradation have historically been the dominant methods for unraveling the mechanisms of protein destruction. The study of protein abundance alterations and protein modifications, a cornerstone of protein degradation pathways, naturally lends itself to global and systematic proteomic methods to decipher the emerging complexity of protein degradation pathways. Advances in proteomic technologies have fueled an explosion of systematic and quantitative studies aimed at understanding how the proteome is shaped and regulated by ubiquitin-dependent processes. These types of studies, as well as targeted analyses of cellular pathways, have revealed that alterations in protein degradation function can have a severe impact on human health and disease. The fusion of these two themes was the focus of the January 2012 conference on proteomics of protein degradation and ubiquitin pathways (PPDUP) held in San Diego. To gain insights into both the current state-of-the-art proteomic methods to investigate protein turnover, and how protein degradation function is altered within a range of human disorders a variety of speakers revealed the many connections between altered protein degradation function and human disease. Many of the sessions were framed by a consistent focus aimed at the discovery and development of novel therapeutics targeting protein degradation pathway components to treat various human maladies ranging from cancer to heart disease.