Inhibition of the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl- ʟ, ʟ-diaminopimelic Acid Desuccinylase from \u3cem\u3eNeisseria meningitidis\u3c/em\u3e by ʟ-Captopril

Binding of the competitive inhibitor ʟ-captopril to the dapE-encoded N-succinyl-ʟ, ʟ-diaminopimelic acid desuccinylase from Neisseria meningitidis (NmDapE) was examined by kinetic, spectroscopic, and crystallographic methods. ʟ-Captopril, an angiotensin-converting enzyme (ACE) inhibitor, was previously shown to be a potent inhibitor of the DapE from Haemophilus influenzae (HiDapE) with an IC50 of 3.3 μM and a measured Ki of 1.8 μM and displayed a dose-responsive antibiotic activity toward Escherichia coli. ʟ-Captopril is also a competitive inhibitor of NmDapE with a Ki of 2.8 μM. To examine the nature of the interaction of ʟ-captopril with the dinuclear active site of DapE, we have obtained electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) data for the enzymatically hyperactive Co(II)-substituted forms of both HiDapE and NmDapE. EPR and MCD data indicate that the two Co(II) ions in DapE are antiferromagnetically coupled, yielding an S = 0 ground state, and suggest a thiolate bridge between the two metal ions. Verification of a thiolate-bridged dinuclear complex was obtained by determining the three-dimensional X-ray crystal structure of NmDapE in complex with ʟ-captopril at 1.8 Å resolution. Combination of these data provides new insights into binding of ʟ-captopril to the active site of DapE enzymes as well as important inhibitor–active site residue interaction’s. Such information is critical for the design of new, potent inhibitors of DapE enzymes

The protein organization of a red blood cell

Red blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-like compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.Cellular and Molecular Biolog

Alternative proteoforms and proteoform-dependent assemblies in humans and plants

Abstract The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing goal in biology. Here, we systematically survey human and plant high-throughput bottom-up native proteomics data for protein truncation variants, where substantial regions of the full-length protein are missing from an observed protein product. In humans, Arabidopsis, and the green alga Chlamydomonas, approximately one percent of observed proteins show a short form, which we can assign by comparison to RNA isoforms as either likely deriving from transcript-directed processes or limited proteolysis. While some detected protein fragments align with known splice forms and protein cleavage events, multiple examples are previously undescribed, such as our observation of fibrocystin proteolysis and nuclear translocation in a green alga. We find that truncations occur almost entirely between structured protein domains, even when short forms are derived from transcript variants. Intriguingly, multiple endogenous protein truncations of phase-separating translational proteins resemble cleaved proteoforms produced by enteroviruses during infection. Some truncated proteins are also observed in both humans and plants, suggesting that they date to the last eukaryotic common ancestor. Finally, we describe novel proteoform-specific protein complexes, where the loss of a domain may accompany complex formation

APP-Induced Patterned Neurodegeneration Is Exacerbated by APOE4 in Caenorhabditis elegans

Genetic and epidemiological studies have found that variations in the amyloid precursor protein (APP) and the apoliopoprotein E (APOE) genes represent major modifiers of the progressive neurodegeneration in Alzheimer’s disease (AD). An extra copy of or gain-of-function mutations in APP correlate with early onset AD. Compared to the other variants (APOE2 and APOE3), the ε4 allele of APOE (APOE4) hastens and exacerbates early and late onset forms of AD. Convenient in vivo models to study how APP and APOE4 interact at the cellular and molecular level to influence neurodegeneration are lacking. Here, we show that the nematode C. elegans can model important aspects of AD including age-related, patterned neurodegeneration that is exacerbated by APOE4. Specifically, we found that APOE4, but not APOE3, acts with APP to hasten and expand the pattern of cholinergic neurodegeneration caused by APP. Molecular mechanisms underlying how APP and APOE4 synergize to kill some neurons while leaving others unaffected may be uncovered using this convenient worm model of neurodegeneration

Inhibition of the <i>dapE</i>-Encoded <i>N</i>‑Succinyl‑l,l‑diaminopimelic Acid Desuccinylase from <i>Neisseria meningitidis</i> by l‑Captopril

Binding of the competitive inhibitor l-captopril to the <i>dapE</i>-encoded <i>N</i>-succinyl-l,l-diaminopimelic acid desuccinylase from <i>Neisseria meningitidis</i> (<i>Nm</i>DapE) was examined by kinetic, spectroscopic, and crystallographic methods. l-Captopril, an angiotensin-converting enzyme (ACE) inhibitor, was previously shown to be a potent inhibitor of the DapE from <i>Haemophilus influenzae</i> (<i>Hi</i>DapE) with an IC₅₀ of 3.3 μM and a measured <i>K</i>_i of 1.8 μM and displayed a dose-responsive antibiotic activity toward <i>Escherichia coli</i>. l-Captopril is also a competitive inhibitor of <i>Nm</i>DapE with a <i>K</i>_i of 2.8 μM. To examine the nature of the interaction of l-captopril with the dinuclear active site of DapE, we have obtained electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) data for the enzymatically hyperactive Co­(II)-substituted forms of both <i>Hi</i>DapE and <i>Nm</i>DapE. EPR and MCD data indicate that the two Co­(II) ions in DapE are antiferromagnetically coupled, yielding an <i>S</i> = 0 ground state, and suggest a thiolate bridge between the two metal ions. Verification of a thiolate-bridged dinuclear complex was obtained by determining the three-dimensional X-ray crystal structure of <i>Nm</i>DapE in complex with l-captopril at 1.8 Å resolution. Combination of these data provides new insights into binding of l-captopril to the active site of DapE enzymes as well as important inhibitor–active site residue interaction’s. Such information is critical for the design of new, potent inhibitors of DapE enzymes

The protein organization of a red blood cell

Red blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-link compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research

High-Content Microfluidic Screening Platform Used To Identify σ2R/Tmem97 Binding Ligands that Reduce Age-Dependent Neurodegeneration in <i>C. elegans</i> SC_APP Model

The nematode <i>Caenorhabditis elegans</i>, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently, we developed a single-copy model of amyloid precursor protein (SC_APP) induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (σ2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure–activity relationships for unexplored chemical space in our σ2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our <i>C. elegans</i> model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that σ2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases