κ-Carrageenase is an important component for κ-carrageenan oligosaccharide production. Generally, noncatalytic domains are appended to carbohydrate-active domains and potentiate catalytic activity. However, studies devoted to κ-carrageenase are relatively few. Here, a C-terminal bacterial immunoglobulin-like domain (Big_2) was identified in κ-carrageenase (PpCgk) from Pseudoalteromonas porphyrae. Biochemical characterization of native PpCgk and its two truncations, PpCgkCD (catalytic domain) and PpBig_2 (Big_2 domain), revealed that the specific activity, catalytic efficiency (k cat /K m(app) ), specific κ-carrageenan-binding capacity, and thermostability of PpCgk were significantly higher than those of PpCgkCD, suggesting that the noncatalytic PpBig_2 domain is a multifunctional module and essential for maintaining the activity and thermostability of PpCgk. Furthermore, it was found that the mode of action of PpCgk was more processive on both the dissolved and gelled substrates than that of PpCgkCD, indicating that PpBig_2 contributes to the processivity of PpCgk. Interestingly, PpBig_2 can be used as an independent module to enhance the hydrolysis of κ-carrageenan through its disruptive function. In addition, sequence analysis suggests that Big_2 domains are highly conserved in bacterial κ-carrageenases, implying the universality of their noncatalytic functions. These findings reveal the multifunctional role of the noncatalytic PpBig_2 and will guide future functional analyses and biotechnology applications of Big_2 domains.

Objective: HuR (human antigen R)—an RNA-binding protein—is involved in regulating mRNA stability by binding adenylate-uridylate–rich elements. This study explores the role of HuR in the regulation of smooth muscle contraction and blood pressure. Approach and Results: Vascular HuR SMKO (smooth muscle–specific HuR knockout) mice were generated by crossbreeding HuR flox/flox mice with α-SMA (α-smooth muscle actin)-Cre mice. As compared with CTR (control) mice, HuR SMKO mice showed hypertension and cardiac hypertrophy. HuR levels were decreased in aortas from hypertensive patients and SHRs (spontaneously hypertensive rats), and overexpression of HuR could lower the blood pressure of SHRs. Contractile response to vasoconstrictors was increased in mesenteric artery segments isolated from HuR SMKO mice. The functional abnormalities in HuR SMKO mice were attributed to decreased mRNA and protein levels of RGS (regulator of G-protein signaling) protein(s) RGS2, RGS4, and RGS5, which resulted in increased intracellular calcium increase. Consistently, the degree of intracellular calcium ion increase in HuR-deficient smooth muscle cells was reduced by overexpression of RGS2, RGS4, or RGS5. Finally, administration of RGS2 and RGS5 reversed the elevated blood pressure in HuR SMKO mice. Conclusions: Our findings indicate that HuR regulates vascular smooth muscle contraction and maintains blood pressure by modulating RGS expression.

The release of neuropeptides from dense core vesicles (DCVs) modulates neuronal activity and plays a critical role in cognitive function and emotion. The granin family is considered a master regulator of DCV biogenesis and the release of DCV cargo molecules. The expression of the VGF protein (nonacronymic), a secreted neuropeptide precursor that also belongs to the extended granin family, has been previously shown to be induced in the brain by hippocampus-dependent learning, and its downregulation is mechanistically linked to neurodegenerative diseases such as Alzheimer’s disease and other mood disorders. Currently, whether changes in translational efficiency of Vgf and other granin mRNAs may be associated and regulated with learning associated neural activity remains largely unknown. Here, we show that either contextual fear memory training or the administration of TLQP-62, a peptide derived from the C-terminal region of the VGF precursor, acutely increases the translation of VGF and other granin proteins, such as CgB and Scg2, via an mTOR-dependent signaling pathway in the absence of measurable increases in mRNA expression. Luciferase-based reporter assays confirmed that the 3′-untranslated region (3′UTR) of the Vgf mRNA represses VGF translation. Consistently, the truncation of the endogenous Vgf mRNA 3′UTR results in substantial increases in VGF protein expression both in cultured primary neurons and in brain tissues from knock in mice expressing a 3′UTR-truncation mutant encoded by the modified Vgf gene. Importantly, Vgf 3′UTR-truncated mice exhibit enhanced memory performance and reduced anxiety- and depression-like behaviors. Our results therefore reveal a rapid, transcription-independent induction of VGF and other granin proteins after learning that are triggered by the VGF-derived peptide TLQP-62. Our findings suggest that the rapid, positive feedforward increase in the synthesis of granin family proteins might be a general mechanism to replenish DCV cargo molecules that have been released in response to neuronal activation and is crucial for memory function and mood stability.

Background Radiation-induced brain injury (RIBI) is the most serious complication of radiotherapy in patients with head and neck tumors, which seriously affects the quality of life. Currently, there is no effective treatment for patients with RIBI, and identifying new treatment that targets the pathological mechanisms of RIBI is urgently needed. Methods Immunofluorescence staining, western blotting, quantitative real-time polymerase chain reaction (Q-PCR), co-culture of primary neurons and microglia, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, enzyme-linked immunosorbent assay (ELISA), and CRISPR–Cas9-mediated gene editing techniques were employed to investigate the protective effects and underlying mechanisms of pregabalin that ameliorate microglial activation and neuronal injury in the RIBI mouse model. Results Our findings showed that pregabalin effectively repressed microglial activation, thereby reducing neuronal damage in the RIBI mouse model. Pregabalin mitigated inflammatory responses by directly inhibiting cytoplasmic translocation of high-mobility group box 1 (HMGB1), a pivotal protein released by irradiated neurons which induced subsequent activation of microglia and inflammatory cytokine expression. Knocking out neuronal HMGB1 or microglial TLR2/TLR4/RAGE by CRISPR/Cas9 technique significantly inhibited radiation-induced NF-κB activation and pro-inflammatory transition of microglia. Conclusions Our findings indicate the protective mechanism of pregabalin in mitigating microglial activation and neuronal injury in RIBI. It also provides a therapeutic strategy by targeting HMGB1-TLR2/TLR4/RAGE signaling pathway in the microglia for the treatment of RIBI.