Jia Liu

Background: Olfactomedin-like protein 3 (OLFML3) is a secreted glycoprotein that is primarily deemed to be associated with embryonic development, angiogenesis, and tumorigenesis. Recent studies have highlighted the function of OLFML3 as an important regulatory protein in viral and bacterial infections. This study explores the role of OLFML3 in type I interferon (IFN-I) signaling in RNA virus infection and related mechanism of action. Methods: RT-qPCR was used to determine the effects of OLFML3 on IFN-I production in the RNA virus infection. Olfml3 ^-/- mice was used to evaluate the effects of OLFML3 in in vivo viral infection. Western blotting (WB) and immunofluorescence microscropy experiments were conducted to analyze the effects of OLFML3 on retinoic-acid-inducible gene I (RIGI)-mediated IFN-I signaling pathway. Mass spectrometry and co-immunoprecipitation (Co-IP) were used to analyze the partner proteins of OLFML3. Gain-of-function studies by overexpression and loss-of-function studies by gene knockout and knockdown were performed to understand the functions of OLFML3 and TRIM21 in RIG-I-mediated IFN-I signaling pathway. Results: OLFML3 can inhibit IFN-I production in RNA virus infection by suppressing RIG-I signaling. OLFML3 interacts with the PRY/SPRY domain of TRIM21, an E3 ubiquitinligase. OLFML3 can disrupt TRIM21-mediated RIG-I K63-ubiquitination, leading to the destabilization of RIG-I and suppressed IFN-I signaling. Conclusion: OLFML3 functions as a general immunosuppressor in RNA virus infection. These results may help developing OLFML3-targeted antiviral therapeutics for the re-activation of IFN-I signaling.

Skeletal muscle is the largest metabolic demanding organ in human body. Alterations of skeletal muscle in shape and size significantly affect its biological functions. Botulinum neurotoxin type A1 (BoNT/A1) has been successfully used in clinics to treat masseter, trapezius and gastrocnemius hypertrophy. Here, we used a healthy rat-based skeletal muscle hypertrophy model to evaluate the muscle-reducing activity of recombinant BoNT/A1 (rBoNT/A1) with genetically fused cell-penetrating peptides (CPPs), which was previously reported to increase the cellular uptake of BoNT/A1. Analyses of treated muscle sections using hematoxylin–eosin and immunofluorescence staining showed that both wild-type rBoNT/A1 without modification (WT-rBoNT/A1) and rBoNT/A1 with CPP fusion (CPP-rBoNT/A1) could induce myocomma atrophy and altered gastrocnemius muscle fiber proportions as a result of denervation and reinnervation. Importantly, rBoNT/A1 with the fusion of a specific CPP, zinc finger protein (ZFP), resulted in the highest degree of muscle atrophy and greatest increase in the ratio of type I muscle fibers over type II fibers. An examination of gastrocnemius muscle cells at the subcellular levels using TEM staining revealed swelled mitochondria and diminished mitochondrial crista upon rBoNT/A1 administration. Transcriptomic RNA sequencing (RNA-Seq) analysis followed by RT-qPCR validation showed that rBoNT/A1 treatment also caused changes in mitochondrial biogenesis and mitophagy. Collectively, our results demonstrated that rBoNT/A1 proteins could alleviate skeletal muscle hypertrophy, with associated alterations of mitochondrial homeostasis.

Protein conformational changes drive signal transduction to regulate cellular activities, yet monitoring of these changes in living cells remains challenging. Here, we introduce BIOSCE (BIOprobe based on Steric Confinement-induced Emission), a technique that enables tracking of individual protein conformations in living cells across millisecond-to-minute timescales. BIOSCE reports protein conformational changes via steric confinement–induced luminescence switching from non-luminescent to luminescent states. We demonstrate that BIOSCE rapidly senses calmodulin conformational changes triggered by intracellular calcium fluctuations. The BIOSCE platform achieved millisecond-resolution monitoring of single-protein conformations within cellular signaling pathways, as evidenced by its sensitive detection of rapamycin-dependent FKBP (FK506-binding protein)-FRB (FKBP-rapamycin binding) interactions regardless of the labeled partner. Furthermore, we applied BIOSCE to track the spatial distribution of SNAP25 (25 kDa synaptosomal nerve-associated protein) during botulinum neurotoxin A (BoNT/A) intoxication, revealing differential catalytic processing of its cleavage fragments. This generalizable approach provides a robust platform for investigating single-molecule conformational changes with high spatiotemporal resolution and enables direct evaluation of transient cellular events.

Cas12a is one of the most widely used Cas nucleases for genome editing and in vitro diagnosis. A number of engineered Cas12a mutants have been identified with improved activity and stability. However, it remains largely unaddressed how these mutations interact. In a previous study, we used a deep learning model to evolve the stability of Lachnospiraceae bacterium Cas12a (LbCas12a) and obtained about 90 mutants with improved stability. In the present study, we performed a deep analysis of these stabilizing mutations and mutation combinations to understand the stability landscape of LbCas12a. It was found that most of the stabilized mutants had shifted fitness, as characterized by higher trans‐cleavage activity at high temperatures but lower activity at the “fit” temperature for the parent protein. These stabilizing mutations were found to have sophisticated epistatic effects. Stabilizing mutation S962K improved protein stability in the context of other stabilizing mutations but by itself exhibited minor improvements. Saturation mutagenesis of S962 had differential effects on the stability of wild‐type (WT) LbCas12a and C10L/I976L/C1090D variant, despite similar melting temperatures (T_m) for WT (41.9°C) and C10L/I976L/C1090D (41.1°C). Interestingly, 12 out of 19 amino acid substitutions at S962 reduced the T_m in the context of WT LbCas12a, while 18 out of 19 mutations increased T_m in the C10L/I976L/C1090D variant. We also showed that stabilizing mutations could recover the stability and trans‐activity of a destabilized LbCas12a variant. Our findings can facilitate the understanding of LbCas12a natural evolution and provide insights to developing novel engineering strategies for Cas nucleases.

Olfactomedin-like protein 3 (OLFML3), belonging to olfactomedin (OLF) protein family, has poorly defined functions. Recent studies have reported the functions of OLFML3 in anti-viral immunity and tumorigenesis. In this study, we investigated the roles of OLFML3 in macrophages. In LPS- or Pseudomonas aeruginosa-induced acute lung injury (ALI) mouse model, OLFML3 depletion exacerbated inflammatory response, leading to reduced survival. OLFML3 achieved the in vivo activity by regulating macrophage phagocytosis and migration. Mass spectrometry analysis revealed immunoresponsive gene 1 (IRG1) as an OLFML3-interacting protein. IRG1 is a mitochondrial decarboxylase that catalyzes the conversion of cis-aconitate to itaconate, a myeloid-borne mitochondrial metabolite with immunomodulatory activities. Further investigation showed that OLFML3 could prevent LPS-induced mitochondrial dysfunction in macrophages by maintaining the homeostasis of mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (mtROS) and itaconate-related metabolites. In-depth protein-protein interaction studies showed that OLFML3 could promote IRG1 mitochondrial localization via a mitochondrial transport protein, apoptosis inducing factor mitochondria associated 1 (AIFM1). In summary, our study showed that OLFML3 could facilitate IRG1 mitochondrial localization and prevent LPS-induced mitochondrial dysfunction in macrophages.

Despite the tremendous success of combination antiretroviral therapy (ART) to treat human immunodeficiency virus (HIV) infection, the durability and persistence of latent reservoirs of HIV-infected cells in HIV-infected patients remain obstacles to achieving HIV cure. While technically challenging, the most direct means to eradicate latent reservoirs is to destroy the HIV provirus, thus ensuring that HIV virions are not produced while preserving resident cells. Transcription activator-like effector nucleases (TALEN)─a genome editing method with high DNA targeting efficiency─have been investigated as a potential gene therapy by disrupting the HIV-1 coreceptor CCR5 genes in HIV target cells or HIV proviral DNA in infected cells. However, the transduction and editing efficiencies are low in primary cells and vary by cell type. Using a nanotechnology platform, which we term nanocapsules, the TALEN protein can be effectively delivered into primary cells and escape from endosome/lysosome sequestration. We report that TALEN nanocapsules can effectively mutagenize the HIV-1 proviral DNA integrated into two primary HIV-1 reservoir cells─T cells and macrophages, such that replication and/or reactivation from latency is aborted. We envision that this study provides a useful platform to deliver a wide range of DNA-modifying enzymes for effective HIV therapy.

Human adenovirus (HAdV) is a widely spread respiratory pathogen that can cause infections in multiple tissues and organs. Previous studies have established an association between HAdV species B (HAdV-B) infection and severe community-acquired pneumonia (SCAP). However, the connection between SCAP-associated HAdV-B infection and host factor expression profile in patients has not been systematically investigated. Here, we perform a CRISPR genetic screen on HAdV-B using two generations of cell surface protein-focused CRISPR libraries and identify a series of host factors including the known receptor DSG-2 and an unknown factor, activated leukocyte cell adhesion molecule (ALCAM). Further investigation shows that ALCAM affects HAdV-B infection by participating in viral internalization. Transcriptomics data from human blood samples suggests that ALCAM expression is higher in SCAP patients with HAdV-B infection than in those with other infections. Chimeric and authentic virus experiments show that ALCAM is a widely used host factor across B1 and B2 genetic clusters of HAdV-B. The dissociation constant between the knob domain of HAdV-B fiber and ALCAM is 837 nM in average. In summary, our results suggest that ALCAM is an entry factor for SCAP-associated HAdV-B.