Protein Research Foundation

Humoral factors regulating cardiac myocytes have not yet been fully elucidated. Using mass spectrometry, we analyzed peptides released from primary neonatal rat cardiac fibroblasts to identify a 24-amino-acid peptide with an intramolecular disulfide bond. It is derived from the C-terminal end of integral membrane protein 2A (ITM2A), a single-pass type II membrane protein; the peptide sequence is identical between humans and rodents and N-terminally flanked by conserved dibasic residues KR. The ITM2A peptide stimulated secretion of atrial natriuretic peptide (ANP), the predominant hormone produced by cardiac myocytes, in a Langendorff rat heart perfusion system as well as a primary culture of neonatal rat ventricular myocytes. These ANP secretion changes were not accompanied by an alteration in the beating rate, a key determinant of ANP secretion. In a mouse cardiac infarction model, both plasma ANP concentration and ITM2A gene expression in heart tissue reached a peak level on day 7 after operation. Our findings provide new insight into cardiac cell-to-cell communication that regulates ANP secretion.

Three-dimensional covalent organic frameworks (3D COFs) are promising crystalline porous materials, but the elucidation of their structure remains challenging, particularly for those featuring spiroborate linkages. Herein, we report the synthesis of a 3D single-crystalline COF (scCOF) with nbo topology, constructed from a rigid square-planar monomer, tetracyclopentatetraphenylene (TCTP), and spiroborate linkages. Theoretical calculations revealed that the TCTP core possesses higher rigidity than phthalocyanine, effectively suppressing structural fluctuations during framework formation. The structure of the resulting TCTP-COF was successfully determined using microcrystal electron diffraction (MicroED), revealing a non-interpenetrated cubic framework with space group Im-3m. TCTP-COF exhibits high crystallinity, thermal stability up to 320 °C, and permanent porosity with a Brunauer–Emmett–Teller surface area of 1360 m2·g–1. To the best of our knowledge, this work represents the first structural determination of a spiroborate-linked 3D scCOF using electron diffraction methods, providing a new design strategy for expanding the chemical space of highly ordered 3D COF architectures.

Molecular recording is an emerging paradigm for measuring biology over time. Enhancer-mediated genomic recording of activity in multiplex (ENGRAM) is a recently described synthetic biology circuit architecture that converts the transient activity of cis-regulatory elements (CREs) into stable genomic records that can be retrospectively recovered via DNA sequencing. Here we provide a step-by-step protocol for conducting ENGRAM experiments and analyzing the resulting data. We also describe key design considerations for ENGRAM recorders, summarize the strengths and limitations of ENGRAM, and highlight applications, including multiplex signal recording and high-throughput CRE screening. In contrast to other systems for DNA-based recording in mammalian systems, ENGRAM relies on prime editing-mediated insertions to record the activity of a given CRE, such that it is inherently multiplexable-for example, four-base-pair insertions can represent the activities of up to 256 distinct CREs. A further contrast lies with ENGRAM's compatibility with DNA Typewriter, which facilitates the capture of signal order. For users with basic skills in molecular biology, mammalian cell culture and DNA sequencing analysis, ENGRAM experiments can typically be completed within 5-6 weeks.

ABSTRACTSulf1 and Sulf2 are extracellular sulfatases that remove 6‐O‐sulfate from heparan sulfate and thereby regulate cell signaling. Previous studies have revealed that Sulf1/Sulf2 double knockout (KO) mice had defects in differentiation and axon guidance during development, but their functional roles in the adult brain remain largely unknown. We recently found that Sulf1 mRNA is highly expressed in the nucleus accumbens (NAc) shell and that Sulf1 expression is detected in both types of medium spiny neurons expressing dopamine D1 or D2 receptors. Moreover, we found that Sulf1 KO led to changes in membrane excitability and excitatory synaptic transmission in medium spiny neurons of the NAc in adult mice. These findings suggest possible roles of Sulf1 in the functions of NAc circuitry. To address this question, we performed behavioral tests using Sulf1 KO mice. We found that constitutive Sulf1 KO mice showed impairment in both the cocaine‐induced conditioned place preference (CPP) test and inhibitory avoidance (IA) test. Next, to examine which cell types the Sulf1 gene is required for, we generated Sulf1 floxed mice by means of CRISPR‐Cas9‐mediated genome editing and mated them with mice expressing Cre recombinase under a promoter for either the dopamine D1 or D2 receptor‐encoding genes. Sulf1 conditional knockout (cKO) in cells expressing dopamine D1 receptors led to impairment only in the CPP test, whereas Sulf1 cKO in D2 receptor‐expressing cells resulted in impairment only in the IA test. These results demonstrate that Sulf1 is required for both reward and aversion learning, and that the D1‐ and D2‐pathways distinctly regulate these functions. The present study suggests that Sulf1 is essential for neuronal functions and behavioral control in the adult brain.

RTS,S/AS01, an adjuvanted subunit malaria vaccine, induces protective but short-lived anti-Plasmodium falciparum circumsporozoite protein (CSP) antibody titers. To better understand the lack of sustained protection post-vaccination, we analyzed CSP-specific B cells over time in malaria-naive individuals following immunization with RTS,S/AS01 followed by controlled malaria infection. Longitudinal analyses revealed a shift in the repertoire and regional protein specificity of CSP-specific B cells over time. Early post-vaccine responses were dominated by class-switched memory B cells (MBCs) specific for the NANP-repeat region of CSP, epitopes associated with protective antibodies. However, the frequency of NANP-repeat-specific MBCs declines, while longer-term memory responses, including responses both before and after a malaria challenge, had increased frequencies of MBCs specific for the C-terminus of CSP. Despite class-switching and affinity maturation, C-term monoclonal antibodies derived from these MBCs failed to protect mice against a transgenic parasite challenge. Taken together, these findings suggest that RTS,S/AS01 induces a transient, protective NANP-repeat-specific B cell response that is subsequently replaced by B cells with non-protective reactivities, potentially explaining its limited long-term efficacy.

Malaria vaccines provide waning protection from disease that is correlated with the production of antibodies to the repeat region of the liver-stage circumsporozoite protein (CSP). CSP-based vaccines display limited durability in malaria-naïve individuals yet are even less effective in malaria-experienced individuals, suggesting the generation of non-optimal humoral immunity in response to both infection and vaccination. To address this hypothesis, we performed a cross-species, comprehensive analysis of B cell responses to CSP after Plasmodium infection or immunization, focusing our analysis on the repeat and C-terminus domains included in malaria subunit vaccines. Herein we demonstrate that while multiple factors may impinge on strong, lasting protective CSP-specific immunity, two forces predominantly impact proper memory B cell differentiation; a) inhibition of germinal center formation by infection and b) skewed B cell differentiation due to the repetitive nature of the protective region of the CSP protein.

Abstract Protein adsorption at solid–liquid interfaces underlies many biomedical and materials applications, yet the mechanisms governing adsorption of proteins and their assemblies remain poorly understood. Here we investigate de novo–designed proteins that self‐assemble into three fibrillar morphologies — small (S), large (L), and helical (H)—on muscovite mica. S‐fibers are metastable, forming first but diminishing as L‐ and H‐fibers develop and deposit. Adsorption of both fibers and monomers depends on fiber morphology and solvent environment, which is modulated by the substrate. The anisotropic surface features of the fiber types—long axis of S‐ and L‐fibers and helical grooves of H‐fibers—correlate with specific crystallographic directions on mica (001). S‐ and L‐fibers align along the unique lattice axis, while the substrate‐facing groove of H‐fibers preferentially aligns along the remaining symmetry‐related directions. Increasing potassium chloride (KCl) concentrations to molar levels alters adsorption, decreasing monomer coverage relative to fibers. These observations, interpreted through the effect of interfacial interface, indicate that ion‐mediated modulation of hydration layers governs electrostatic interactions and alignment. This study reveals how coupling between protein topography, substrate crystallography, and hydration structure dictates selective adsorption and orientation of protein assemblies, offering insights for rational biomolecular material design.