Choice of fusion proteins, expression host, and analytics solves difficult-to-produce protein challenges in discovery research.

High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein-based discovery research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts-91% of which are successfully expressed and purified-are discussed. Mammalian expression combined with the use of solubility-promoting fusion proteins is deemed suitable for most targets. Furthermore, cases utilizing stable cell line generation and choice of fusion protein for higher yield and quality of difficult-to-produce proteins (Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) and Neurturin) are presented and discussed. In the case of Neurturin, choice of fusion protein impacted the target binding 80-fold. These results highlight the need for exploration of construct designs and careful Quality Control (QC) of difficult-to-produce protein reagents.

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.

The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3ß to foster phosphorylation of the Wnt effector ß-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced ß-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.

Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.

Crystal structure of human aquaporin 4 at 1.8 A and its mechanism of conductance.

Aquaporin (AQP) 4 is the predominant water channel in the mammalian brain, abundantly expressed in the blood-brain and brain-cerebrospinal fluid interfaces of glial cells. Its function in cerebral water balance has implications in neuropathological disorders, including brain edema, stroke, and head injuries. The 1.8-A crystal structure reveals the molecular basis for the water selectivity of the channel. Unlike the case in the structures of water-selective AQPs AqpZ and AQP1, the asparagines of the 2 Asn-Pro-Ala motifs do not hydrogen bond to the same water molecule; instead, they bond to 2 different water molecules in the center of the channel. Molecular dynamics simulations were performed to ask how this observation bears on the proposed mechanisms for how AQPs remain totally insulating to any proton conductance while maintaining a single file of hydrogen bonded water molecules throughout the channel.

Endoscopic antireflux therapy: the Stretta procedure.

The Stretta procedure is safe and effective for the treatment of GERD. There are well-documented clinical trial data supporting its use, including a randomized sham-controlled study, single- and multi-center prospective trials, and community practice reports. The complication rate is within the acceptable range for therapeutic endoscopic procedures and less than the published complication rate for laparoscopic fundoplication. The durability of effect also is established beyond 2 years in several studies. Stretta should be added to the GERD management algorithm specifically for patients considering an antireflux surgical procedure but who are not accepting of the risks of surgery and anesthesia. These patients typically present with incomplete GERD control, despite optimal antisecretory drug therapy, or intolerance to medical therapy. Stretta should be considered only for patients who fit the anatomic inclusion criteria, whereas antireflux surgery should be reserved for those who do not. The decision to undergo antireflux surgery or Stretta must be based on the relative risks and benefits of each procedure. Although antireflux surgery provides better control of esophageal acid exposure than Stretta, the outcomes for GERD symptoms, quality of life, and reduction in PPI use are comparable. Stretta has a low risk of acute adverse events, has no reported cases of long-term dysphagia, and obviates general anesthesia and hospitalization, whereas antireflux surgery has a reported adverse event rate of approximately 2%, a considerable incidence of dysphagia, and requires general anesthesia and 1 to 2 days in the hospital. Another advantage of the Stretta procedure is that antireflux surgery still can be performed in the case of failures. In conclusion, the Stretta procedure offers a minimally invasive, safe, and effective alternative to antireflux surgery for those patients who have GERD who are controlled unsatisfactorily on antisecretory medications, who are considering surgery, and who meet the anatomic criteria that make the procedure technically feasible and safe.

Endoscopic mucosal resection for early cancers of the upper gastrointestinal tract.

The purpose of this literature review is to examine recent advances in technique and technology of endoscopic mucosal resection of superficial early cancers of the upper gastrointestinal tract. Endoscopic mucosal resection (EMR) of superficial early cancers of the upper gastrointestinal tract is standard technique in Japan and is increasingly used in Western countries. Newer techniques of EMR allow removal of larger lesions en-bloc. These minimally invasive techniques, when applied correctly, allow safe and efficacious treatment in situations that would otherwise require major surgery. Through the establishment of long-term outcomes data, standardization of endoscopic and pathologic reporting, and newer EMR technology and techniques, the future treatment of early cancers in the upper gastrointestinal tract may be achieved primarily through the endoscope.

Endoscopic therapy for Barrett's esophagus.

With the increase in the rate of esophageal adenocarcinoma in the United States and the Western world matched with the high morbidity and mortality of esophagectomy, there is an increasing need for new and effective techniques to treat and prevent esophageal adenocarcinoma. A wide variety of endoscopic mucosal ablative techniques have been developed for early esophageal neoplasia. However, long-term control of neoplasic risk has not been demonstrated. Most studies show that specialized intestinal metaplasia may persist underneath neo-squamous mucosa, posing a risk for subsequent neoplastic progression. In this article we review current published literature on endoscopic therapies for the management of Barrett's esophagus.

Baclofen toxicity in an 8-year-old with an intrathecal baclofen pump.

Baclofen delivered by intrathecal pumps (ITB) is increasingly being utilized in the pediatric population, however, resources and education to support problems with these devices are limited. Typical management strategies for systemic baclofen overdose include removal of baclofen from the device reservoir or removal of cerebrospinal fluid from the adjacent device catheter. Appropriate care of these patients requires awareness of the clinical patterns of toxicity and mechanics of the ITB pump delivery system. This report describes the clinical presentation, unfamiliar dilemmas, and the management of a pediatric patient with intrathecal baclofen toxicity, noting problems that may arise in the care of these patients.