Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that functions in many cellular pathways including steroid receptor (SR) coactivation, heterochromatin maintenance, and ribosome biogenesis. PELP1 is a proto-oncogene whose expression is upregulated in many human cancers, but how the PELP1 scaffold coordinates its diverse cellular functions is poorly understood. Here we show that PELP1 serves as the central scaffold for the human Rix1 complex whose members include WDR18, TEX10, and SENP3. We reconstitute the mammalian Rix1 complex and identified a stable sub-complex comprised of the conserved PELP1 Rix1 domain and WDR18. We determine a 2.7 Å cryo-EM structure of the subcomplex revealing an interconnected tetrameric assembly and the architecture of PELP1's signaling motifs, including eleven LxxLL motifs previously implicated in SR signaling and coactivation of Estrogen Receptor alpha (ERα) mediated transcription. However, the structure shows that none of these motifs is in a conformation that would support SR binding. Together this work establishes that PELP1 scaffolds the Rix1 complex, and association with WDR18 may direct PELP1's activity away from SR coactivation.

Association between preoperative gonioscopic status and postoperative glaucoma after phacoemulsification in dogs: A retrospective cohort study of 505 eyes.

OBJECTIVE: To assess whether preoperative gonioscopy status is associated with the development of postoperative glaucoma after phacoemulsification. PROCEDURE: Eligible dogs and eyes were selected from medical records (2007-2017), and each eye was classified as having normal or abnormal gonioscopy status. Records were examined for postoperative glaucoma, and data were collected for baseline variables including patient signalment, surgical factors, and pre, intra, and postoperative medications. RESULTS: Of 305 dogs (505 eyes) selected for review in this study, 111 dogs (163 eyes) had abnormal gonioscopy findings, and 194 dogs (342 eyes) had normal gonioscopy findings. Postoperative glaucoma occurred in 24% (39/163) of eyes with abnormal gonioscopy and in 14% (49/342) eyes with normal gonioscopy. Eyes with abnormal gonioscopy status were at higher risk of postoperative glaucoma compared to normal eyes (P = .025). There was no important confounding due to the numerous baseline variables summarised above. Regardless of gonioscopy status, the risk of glaucoma was highest during the first 2 months after surgery. CONCLUSIONS: Eyes with abnormal gonioscopy findings are at increased risk of postoperative glaucoma compared with eyes with normal gonioscopy findings. Gonioscopy is recommended as a part of presurgical assessment in all dogs prior to phacoemulsification. Further study is needed to assess whether the incidence of postoperative glaucoma can be reduced by increased monitoring or altered treatment protocols in cases that are known to have abnormal gonioscopy findings prior to cataract surgery.

Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis.

AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.

Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7.

Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-α7 insertions within both AAA domains important for Rix7 function.

Structural Analysis Reveals Features of Ribosome Assembly Factor Nsa1/WDR74 Important for Localization and Interaction with Rix7/NVL2.

Ribosome assembly is a complex process that requires hundreds of essential assembly factors, including Rix7 (NVL2 in mammals) and Nsa1 (WDR74 in mammals). Rix7 is a type II double ring, AAA-ATPase, which is closely related to the well-known Cdc48/p97. Previous studies in Saccharomyces cerevisiae suggest that Rix7 mediates the release of Nsa1 from nucleolar pre-60S particles; however, the underlying mechanisms of this release are unknown. Through multiple structural analyses we show that S. cerevisiae Nsa1 is composed of an N-terminal seven-bladed WD40 domain followed by a lysine-rich C terminus that extends away from the WD40 domain and is required for nucleolar localization. Co-immunoprecipitation assays with the mammalian homologs identified a well-conserved interface within WDR74 that is important for its association with NVL2. We further show that WDR74 associates with the D1 AAA domain of NVL2, which represents a novel mode of binding of a substrate with a type II AAA-ATPase.

The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor Ytm1 and Characterization of Its Interaction with the AAA-ATPase Midasin.

The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous non-ribosomal factors, such as the PeBoW complex. The mammalian PeBoW complex, composed of Pes1, Bop1, and WDR12, is essential for the processing of the 32S preribosomal RNA. Previous work in Saccharomyces cerevisiae has shown that release of the homologous proteins in this complex (Nop7, Erb1, and Ytm1, respectively) from preribosomal particles requires Rea1 (midasin or MDN1 in humans), a large dynein-like protein. Midasin contains a C-terminal metal ion-dependent adhesion site (MIDAS) domain that interacts with the N-terminal ubiquitin-like (UBL) domain of Ytm1/WDR12 as well as the UBL domain of Rsa4/Nle1 in a later step in the ribosome maturation pathway. Here we present the crystal structure of the UBL domain of the WDR12 homologue from S. cerevisiae at 1.7 Å resolution and demonstrate that human midasin binds to WDR12 as well as Nle1 through their respective UBL domains. Midasin contains a well conserved extension region upstream of the MIDAS domain required for binding WDR12 and Nle1, and the interaction is dependent upon metal ion coordination because removal of the metal or mutation of residues that coordinate the metal ion diminishes the interaction. Mammalian WDR12 displays prominent nucleolar localization that is dependent upon active ribosomal RNA transcription. Based upon these results, we propose that release of the PeBoW complex and subsequent release of Nle1 by midasin is a well conserved step in the ribosome maturation pathway in both yeast and mammalian cells.

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Conjunctival lymphoma: immunophenotype and outcome in five dogs and three cats.

Conjunctival lymphoma is well documented in the medical literature, but veterinary reports are few. We report five cases of canine lymphoma, and three of feline in which the presenting sign was conjunctival involvement. All animals were in apparently good health at the time of presentation, and attended the referring clinic because of conjunctival disease. One dog showed generalized lymphadenopathy at presentation, although the ocular lesion was the reason for consultation, but all other patients were well with no detectable disease beyond the eye. All cats were presented for their ocular disease. All dogs were confirmed to have T-cell tumors, although the histological appearance of these was variable. In contrast, all cats had B-cell tumors. Referring clinicians and owners were contacted for follow-up information. Three dogs had been euthanased within 6 months of diagnosis for deterioration of general health. The remaining two were alive and showed no signs of systemic disease. Two cats had good survival following diagnosis, the other died of lesions that may not be related.

Surgical management and outcome of lower eyelid entropion in 124 cats.

OBJECTIVES: To evaluate the success rate of various surgical techniques for the management of lower eyelid entropion in cats. DESIGN: Retrospective study. Animals studied One hundred and twenty-four cats with surgical correction of lower eyelid entropion of 200 eyes over a 13 year period. METHODS: Records of 124 cats were reviewed for signalment, type of entropion, surgical procedure performed and post-operative result. RESULTS: Combinations of the Hotz-Celsus (HC), lateral canthal closure and full thickness wedge resection techniques were used to treat 64 bilateral and 60 unilateral cases of lower lid entropion. Twenty-three cats were under a year of age, 52 cats were aged between 2 and 8 years and 49 were over 8 years old. The overall success rate for a single surgical procedure (which may consist of multiple techniques) to correct lower eyelid entropion was 96.0% per eye. The remaining 4.0% had the entropion resolved with a second surgery. A combined HC and lateral canthal closure had a 99.21% success rate of resolving lower lid entropion. Geriatric cats were the most likely age group to develop corneal sequestra; 37% of cats in this group presented with entropion and corneal sequestra concurrently. Seventeen percent of cats that presented with unilateral entropion and did not have prophylactic surgery on the fellow eye went on to develop entropion in the fellow eye. CONCLUSIONS: A combined HC and lateral canthal closure was the most effective surgical technique in managing lower eyelid entropion of cats in our study. Prophylactic lateral canthal closure in the unaffected eye is recommended.

Pigmented squamous cell carcinoma of the conjunctiva of a horse.

Abstract A heavily pigmented tumor was removed from the lateral, perilimbal, bulbar conjunctiva of a bay Thoroughbred horse. Excision was incomplete and the base of the tumor was lasered on the assumption that the tumor was a melanoma. No other ophthalmic lesions were seen. Histology showed the mass to be a pigmented squamous cell carcinoma (SCC). The prognosis and recommended treatment protocols are different for SCC and melanoma. This is, to the best of our knowledge, the first report of an ocular, pigmented SCC in the veterinary literature.

Taenia serialis causing exophthalmos in a pet rabbit.

A 16-month-old, male, neuter Dwarf Lop rabbit presented with exophthalmos of the right eye of 3 weeks duration. Under sedation an ultrasound of the right eye was performed and showed an orbital hypo-echoic area posterior and ventrolateral to the right globe, which was presumed to be a cyst. Fine needle aspirate removed 5.5 mLs of straw-colored fluid from the cyst, which allowed the globe to return to its normal position. Two months later the rabbit re-presented with exophthalmos of the right eye. Exploratory surgery was performed and a large cystic structure was removed from the ventro-lateral conjunctival fornix. Histology confirmed the cyst to be a coenurus of Taenia serialis.

Transitional cell carcinoma metastatic to the eye in a collared peccary (Tayassu tajacu).

A 15-year-old female collared peccary (Tayassu tajacu) was presented for ophthalmic examination following sudden onset of blindness. Bilateral retinal detachment was diagnosed, neoplasia suspected, and euthanasia performed. Widespread tumor dissemination was apparent at autopsy, and transitional cell carcinoma was diagnosed histologically. The tumor was identified as arising from the ovary. Epidemiologic features of this case are discussed.

Marsupial ophthalmology.

This article discusses ocular conditions found in marsupials. Marsupials are unique models for developmental biology because of their immature state of development at birth. There is considerable variation in the ocular evolution of marsupials, largely in response to their unique diversification. Many marsupials and their eyes have been studied.