A high-resolution protein architecture of the budding yeast genome.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.

The CalDAG-GEFI/Rap1/αIIbß3 axis minimally contributes to accelerated platelet clearance in mice with constitutive store-operated calcium entry.

Circulating platelets maintain low cytosolic Ca2+ concentrations. At sites of vascular injury, agonist-induced Ca2+ release from platelet intracellular stores triggers influx of extracellular Ca2+, a process known as store-operated Ca2+ entry (SOCE). Stromal interaction molecule 1 (Stim1) senses reduced Ca2+ stores and triggers SOCE. Gain-of-function (GOF) mutations in Stim1, such as described for Stormorken syndrome patients or mutant mice (Stim1Sax), are associated with marked thrombocytopenia and increased platelet turnover. We hypothesized that reduced platelet survival in Stim1Sax/+ mice is due to increased Rap1/integrin signaling and platelet clearance in the spleen, similar to what we recently described for mice expressing a mutant version of the Rap1-GAP, Rasa3 (Rasa3hlb/hlb). Stim1Sax/+ mice were crossed with mice deficient in CalDAG-GEFI, a critical calcium-regulated Rap1-GEF in platelets. In contrast to Rasa3hlb/hlb x Caldaggef1-/- mice, only a small increase in the peripheral platelet count, but not platelet lifespan, was observed in Stim1Sax/+ x Caldaggef1-/- mice. Similarly, inhibition of αIIbß3 integrin in vivo only minimally raised the peripheral platelet count in Stim1Sax/+ mice. Compared to controls, Stim1Sax/+ mice exhibited increased platelet accumulation in the lung, but not the spleen or liver. These results suggest that CalDAG-GEFI/Rap1/integrin signaling contributes only minimally to accelerated platelet turnover caused by constitutive SOCE.

What do we know? Platelets are small blood cells which act to prevent blood loss, which circulate in a resting state but are rapidly activated upon exposure to ligands at the site of vascular injuryCalcium (Ca²⁺) is critical for platelet activation, especially for activation of integrins which support platelet­platelet interactionsIf platelet activation occurs in circulation, platelets can be prematurely cleared from blood and unable to function in hemostasisDisorders of Ca²⁺ dysregulation such as Stormorken syndrome are associated with reduced platelet counts (thrombocytopenia) and bleedingWhat did we discover? We used a mouse model expressing a mutation causing higher Ca²⁺ levels in cells including platelets (Stim1^Sax), and investigated whether thrombocytopenia is due to stimulation of a specific pathway for integrin activation, mediated by a protein called Rap1 GTPaseWe crossed Stim1^Sax mice with mice lacking an important activator of Rap1, the Ca²⁺-regulated protein CalDAG-GEFI, and saw no major improvement in thrombocytopeniaWe also observed more Stim1^Sax platelets in the lung but not the liver or spleen, in contrast to mice with activation of platelet integrins in circulationWhat is the impact? Our results rule out activation of the CalDAG-GEFI/Rap1/integrin pathway as a major cause of thrombocytopenia in Stim1^Sax miceOur findings help to narrow down potential causes of thrombocytopenia in disorders such as Stormorken syndrome.

Immunohistochemical mapping and transcript expression of the GPA2/GPB5 receptor in tissues of the adult mosquito, Aedes aegypti.

GPA2/GPB5 is a glycoprotein hormone found in most bilateral metazoans including the mosquito, Aedes aegypti. To elucidate physiological roles and functions of GPA2/GPB5, we aim to identify prospective target tissues by examining the tissue- and sex-specific expression profile of its receptor, the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1) in the adult mosquito. Western analyses using a heterologous system with CHO-K1 cells, transiently expressing A. aegypti LGR1, yielded a 112-kDa monomeric band and high-molecular weight multimers, which associated with membrane-protein fractions. Moreover, immunoblot analyses on protein isolated from HEK 293 T cells stably expressing a fusion construct of A. aegypti LGR1-EGFP (LGR1: 105 kDa+EGFP: 27 kDa) yielded a band with a measured molecular weight of 139 kDa that also associated with membrane-protein fractions and upon deglycosylation, migrated as a lower molecular weight band of 132 kDa. Immunocytochemical analysis of HEK 293 T cells stably expressing this fusion construct confirmed EGFP fluorescence and LGR1-like immunoreactivity colocalized primarily to the plasma membrane. Immunohistochemical mapping in adult mosquitoes revealed LGR1-like immunoreactivity is widespread in the alimentary canal. Importantly, LGR1-like immunoreactivity localizes specifically to basolateral regions of epithelia and, in some regions, appeared as punctate intracellular staining, which together indicates a potential role in feeding and/or hydromineral balance. LGR1 transcript expression was also detected in gut regions that exhibited strong LGR1-like immunoreactivity. Interestingly, LGR1 transcript expression and strong LGR1-like immunoreactivity was also identified in reproductive tissues including the testes and ovaries, which together suggests a potential role linked to spermatogenesis and oogenesis in male and female mosquitoes, respectively.

Functional role of the heterodimeric glycoprotein hormone, GPA2/GPB5, and its receptor, LGR1: An invertebrate perspective.

In vertebrates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid-stimulating hormone (TSH) are glycoprotein hormones that play central roles in metabolism, reproduction and development. Recently, a novel heterodimeric glycoprotein hormone, called GPA2/GPB5, was discovered in humans; however, contrary to its vertebrate glycoprotein hormone relatives, the physiological role of GPA2/GPB5 has not yet been fully elucidated in any vertebrate or invertebrate. Moreover, it is unclear as to whether GPA2/GPB5 functions as a heterodimer or as individual GPA2 and GPB5 monomers in these organisms. GPA2- and GPB5-like subunits have been identified or predicted in a wide array of animal phyla including the nematodes, chordates, hemichordates, arthropods, molluscs, echinoderms and annelids. So far, molecular studies on transcript expression of the GPA2/GPB5 subunits and its putative receptor, the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1), suggests this glycoprotein hormone system plays a developmental role and may also function in hydromineral balance in invertebrates. This mini-review summarizes the current state of knowledge on the physiological actions and activity of this evolutionarily ancient heterodimeric glycoprotein hormone with a particular focus on its known functions in the invertebrates.

An Innovative Polymeric Platform for Controlled and Localized Drug Delivery.

Precision medicine aims to optimize pharmacological treatments by considering patients' genetic, phenotypic, and environmental factors, enabling dosages personalized to the individual. To address challenges associated with oral and injectable administration approaches, implantable drug delivery systems have been developed. These systems overcome issues like patient adherence, bioavailability, and first-pass metabolism. Utilizing new combinations of biodegradable polymers, the proposed solution, a Polymeric Controlled Release System (PCRS), allows minimally invasive placement and controlled drug administration over several weeks. This study's objective was to show that the PCRS exhibits a linear biphasic controlled release profile, which would indicate potential as an effective treatment vehicle for cervical malignancies. An injection mold technique was developed for batch manufacturing of devices, and in vitro experiments demonstrated that the device's geometry and surface area could be varied to achieve various drug release profiles. This study's results motivate additional development of the PCRS to treat cervical cancer, as well as other malignancies, such as lung, testicular, and ovarian cancers.

Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation.

Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.

Evaluating the Performance of a Nonelectronic, Versatile Oxygenating Perfusion System across Viscosities Representative of Clinical Perfusion Solutions Used for Organ Preservation.

Introduction: On the United States' Organ Transplantation Waitlist, approximately 17 people die each day waiting for an organ. The situation continues to deteriorate as the discrepancy between harvested organs and the number of patients in need is increasing. Static cold storage is the clinical standard method for preserving a harvested organ but is associated with several drawbacks. Machine perfusion of an organ has been shown to improve preservation quality as well as preservation time over static cold storage. While there are machine perfusion devices clinically available, they are costly and limited to specific organs and preservation solutions. This study presents a versatile oxygenating perfusion system (VOPS) that supplies oxygen and pulsatile perfusion. Materials and Methods: Experiments evaluated the system's performance with a human kidney mimicking hydraulic analog using multiple compressed oxygen supply pressures and aqueous solutions with viscosities ranging from 1 to 6.5 cP, which simulated viscosities of commonly used organ preservation solutions. Results and Conclusions: The VOPS produced mean flow rates ranging from 0.6 to 28.2 mL/min and perfusion pressures from 4.8 to 96.8 mmHg, which successfully achieved the desired perfusion parameters for human kidneys. This work provides evidence that the VOPS described herein has the versatility to perfuse organs using many of the clinically available preservation solutions.

A genetic model for in vivo proximity labelling of the mammalian secretome.

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Proteomics of protein trafficking by in vivo tissue-specific labeling.

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

Expression Profiling, Downstream Signaling, and Inter-subunit Interactions of GPA2/GPB5 in the Adult Mosquito Aedes aegypti.

GPA2/GPB5 and its receptor constitute a glycoprotein hormone-signaling system native to the genomes of most vertebrate and invertebrate organisms. Unlike the well-studied gonadotropins and thyrotropin, the exact function of GPA2/GPB5 remains elusive, and whether it elicits its functions as heterodimers, homodimers or as independent monomers remains unclear. Here, the glycoprotein hormone signaling system was investigated in adult mosquitoes, where GPA2 and GPB5 subunit expression was mapped and modes of its signaling were characterized. In adult Aedes aegypti mosquitoes, GPA2 and GPB5 transcripts co-localized to bilateral pairs of neuroendocrine cells, positioned within the first five abdominal ganglia of the central nervous system. Unlike GPA2/GPB5 homologs in human and fly, GPA2/GPB5 subunits in A. aegypti lacked evidence of heterodimerization. Rather, cross-linking analysis to determine subunit interactions revealed A. aegypti GPA2 and GPB5 subunits may form homodimers, although treatments with independent subunits did not demonstrate receptor activity. Since mosquito GPA2/GPB5 heterodimers were not evident by heterologous expression, a tethered fusion construct was generated for expression of the subunits as a single polypeptide chain to mimic heterodimer formation. Our findings revealed A. aegypti LGR1 elicited constitutive activity with elevated levels of cAMP. However, upon treatment with recombinant tethered GPA2/GPB5, an inhibitory G protein (Gi/o) signaling cascade is initiated and forskolin-induced cAMP production is inhibited. These results further support the notion that heterodimerization is a requirement for glycoprotein hormone receptor activation and provide novel insight to how signaling is achieved for GPA2/GPB5, an evolutionary ancient neurohormone.

Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti.

Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult Aedes aegypti mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in A. aegypti indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector.