Tandem duplication of serpin genes yields functional variation and snake venom inhibitors.

Tandem duplication of genes can play a critical role in the evolution of functional novelty, but our understanding is limited concerning gene duplication's role in coevolution between species. Much is known about the evolution and function of tandemly duplicated snake venom genes, however the potential of gene duplication to fuel venom resistance within prey species is poorly understood. In this study, we characterize patterns of gene duplication of the SERPINA subfamily of genes across in vertebrates and experimentally characterize functional variation in the SERPINA3-like paralogs of a wild rodent.

High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases.

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue-and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis.

Functional overlap between the mammalian and paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two paralogs, and .

Functional overlap between the mammalian and paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two paralogs, and .

Identification of LMAN1- and SURF4-Dependent Secretory Cargoes.

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the or gene.

An Unbiased Screen Identified the Hsp70-BAG3 Complex as a Regulator of Myosin-Binding Protein C3.

Variants in the gene myosin-binding protein C3 () account for approximately 50% of familial hypertrophic cardiomyopathy (HCM), leading to reduced levels of myosin-binding protein C3 (MyBP-C), the protein product made by gene . Elucidation of the pathways that regulate MyBP-C protein homeostasis could uncover new therapeutic strategies. Toward this goal, we screened a library of 2,426 bioactive compounds and identified JG98, an allosteric modulator of heat shock protein 70 that inhibits interaction with Bcl-2-associated athanogene (BAG) domain co-chaperones.

Identification of LMAN1 and SURF4 dependent secretory cargoes.

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles/tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the or gene.

Cargo selection in endoplasmic reticulum-to-Golgi transport and relevant diseases.

Most proteins destined for the extracellular space or various intracellular compartments must traverse the intracellular secretory pathway. The first step is the recruitment and transport of cargoes from the endoplasmic reticulum (ER) lumen to the Golgi apparatus by coat protein complex II (COPII), consisting of five core proteins. Additional ER transmembrane proteins that aid cargo recruitment are referred to as cargo receptors.

Gradual increase in sweat chloride concentration is associated with a higher risk of CRMS/CFSPID to CF reclassification.

Objectives: Universal implementation of cystic fibrosis (CF) newborn screening (NBS) has led to the diagnostic dilemma of infants with CF screen-positive, inconclusive diagnosis (CFSPID), with limited guidance regarding prognosis and standardized care. Rates of reclassification from CFSPID to CF vary and risk factors for reclassification are not well established. We investigated whether clinical characteristics are associated with the risk of reclassification from CFSPID to a CF diagnosis.

High rates of anxiety detected in mothers of children with inconclusive cystic fibrosis screening results.

Objective: The purpose was to assess postpartum depression, anxiety, and depression in mothers of children with an inconclusive diagnosis after a positive cystic fibrosis (CF) newborn screening (NBS), known as cystic fibrosis transmembrane conductance regulator (CFTR)-related metabolic syndrome (CRMS) or CF screen positive, inconclusive diagnosis (CFSPID). There is limited information on the prognosis and on the impact of this designation on maternal mental health.

Methods: Mothers of children with CRMS/CFSPID and CF identified by NBS were recruited from two centers in California.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C.

Hepatic inactivation of murine results in marked reduction in plasma cholesterol.

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018).

Identification of secreted proteins by comparison of protein abundance in conditioned media and cell lysates.

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach.

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes.

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane.

Identification of novel γ-globin inducers among all potential erythroid druggable targets.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals.

When CFSPID becomes CF.

There has been a growing number of infants identified as CRMS/CFSPID in countries applying genetic testing as part of cystic fibrosis (CF) newborn screening. Currently there are neither standardized protocols for follow up beyond infancy, nor established predictors to stratify this population as high or low risk of reclassification to CF or CFTR-related disorder. We report a series of 10 children who reclassified, including eight carrying CFTR variants of varying clinical consequence and seven with initial sweat chloride measurements <30 mmol/L.

Murine SEC24D can substitute functionally for SEC24C during embryonic development.

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.

Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape.

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system.

The Report of the 2020-2021 Professional Affairs Standing Committee: Pharmacists' Unique Role and Integration in Healthcare Settings.

The 2020-21 Professional Affairs Committee was charged to (1) Read all six reports from the 2019-20 AACP standing committees to identify elements of these reports that are relevant to the committee's work this year; (2) Identify opportunities and models of integration of pharmacist care services in physician and other health provider practices beyond primary care; (3) Differentiate and make the case for the integration of pharmacist care services from that of other mid-level providers; and (4) From the work on the aforementioned charges, identify salient activities for the Center To Accelerate Pharmacy Practice Transformation and Academic Innovation (CTAP) for consideration by the AACP Strategic Planning Committee and AACP staff. This report provides information on the committee's process to address the committee charges, describes the rationale for and the results from a call to colleges and schools of pharmacy to provide information on their integrating pharmacist care services in physician and other health provider practices beyond primary care practice, and discusses how pharmacist-provided patient care services differ from those provided by other healthcare providers. The committee offers a revision to a current association policy statement, a proposed policy statement as well as recommendations to CTAP and AACP and suggestions to colleges and schools of pharmacy pertaining to the committee charges.

Pharmacists Are Not Mid-Level Providers.

Pharmacists should not be classified as "mid-level" providers. This classification implies that there are different levels or a hierarchy of providers when in fact each health care provider brings unique and essential knowledge and contributions to the health care team and to the care of patients. Pharmacists are no exception.

Phage display broadly identifies inhibitor-reactive regions in von Willebrand factor.

Background: Correction of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti-VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD).

IgV somatic mutation of human anti-SARS-CoV-2 monoclonal antibodies governs neutralization and breadth of reactivity.

Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we report a possibly novel approach to rapid generation of potent broadly neutralizing human anti-SARS-CoV-2 Abs.