The role of E3 ubiquitin ligases in bone homeostasis and related diseases

The ubiquitin-proteasome system(UPS)dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms.These enzymatic cascades mark and modifies target proteins diversly through covalently binding ubiquitin molecules.In the UPS,E3 ubiquitin ligases are the crucial constituents by the advantage of recognizing and presenting proteins to proteasomes for proteolysis.As the major regulators of protein homeostasis,E3 ligases are indispensable to proper cell manners in diverse systems,and they are well described in physiological bone growth and bone metabolism.Pathologically,classic bone-related diseases such as metabolic bone diseases,arthritis,bone neoplasms and bone metastasis of the tumor,etc.,were also depicted in a UPS-dependent manner.Therefore,skeletal system is versatilely regulated by UPS and it is worthy to summarize the underlying mechanism.Furthermore,based on the current status of treatment,normal or pathological osteogenesis and tumorigenesis elaborated in this review highlight the clinical significance of UPS research.As a strategy possibly remedies the limitations of UPS treatment,emerging PROTAC was described comprehensively to illustrate its potential in clinical application.Altogether,the purpose of this review aims to provide more evidence for exploiting novel therapeutic strategies based on UPS for bone associated diseases.

An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing.However,natural nucleases of this system often exhibit low efficiency,limiting their application.Here,we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease,Cas12i3.As a result,we developed Cas-SF01,a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells.Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases.Compared to natural Cas12i3,Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs.In addition,we identified an amino acid substitution,D876R,that markedly reduced the off-target effect while maintaining high on-target activity,leading to the development of CasSF01^(HiFi)(high-fidelity Cas-SF01).Finally,we show that Cas-SF01 has high gene editing activities in mice and plants.Our results suggest that CasSF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections.The strong capacity of P.aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure.The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts.Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs.In this study,a highthroughput transposon-insertion site sequencing(Tn-seq)approach was employed to identify novel P.aeruginosa biofilm genetic determinants.When analyzing the novel biofilm regulatory genes,we found that the cell division factor ZapE(PA4438)controls the P.aeruginosa pqs quorum sensing system.The ΔzapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone(PQS)had been reduced.Further biochemical analysis showed that ZapE interacts with PqsH,which encodes the synthase that converts 2-heptyl-4-quinolone(HHQ)to PQS.In addition,site-directed mutagenesis of the ATPase active site of ZapE(K72A)abolished the positive regulation of ZapE on PQS signaling.As ZapE is highly conserved among the Pseudomonas group,our study suggests that it is a potential drug target for the control of Pseudomonas infections.

Targeting Kindlin-2 in adipocytes increases bone mass through inhibiting FAS/PPARγ/FABP4 signaling in mice

Osteoporosis(OP)is a systemic skeletal disease that primarily affects the elderly population,which greatly increases the risk of fractures.Here we report that Kindlin-2 expression in adipose tissue increases during aging and high-fat diet fed and is accompanied by decreased bone mass.Kindlin-2 specific deletion(K2KO)controlled by Adipoq-Cre mice or adipose tissue-targeting AAV(AAV-Rec2-CasRx-sgK2)significantly increases bone mass.Mechanistically,Kindlin-2 promotes peroxisome proliferator-activated receptor gamma(PPARγ)activation and downstream fatty acid binding protein 4(FABP4)expression through stabilizing fatty acid synthase(FAS),and increased FABP4 inhibits insulin expression and decreases bone mass.Kindlin-2 inhibition results in accelerated FAS degradation,decreased PPARγactivation and FABP4 expression,and therefore increased insulin expression and bone mass.Interestingly,we find that FABP4 is increased while insulin is decreased in serum of OP patients.Increased FABP4 expression through PPARγactivation by rosiglitazone reverses the high bone mass phenotype of K2KO mice.Inhibition of FAS by C75 phenocopies the high bone mass phenotype of K2KO mice.Collectively,our study establishes a novel Kindlin-2/FAS/PPARγ/FABP4/insulin axis in adipose tissue modulating bone mass and strongly indicates that FAS and Kindlin-2 are new potential targets and C75 or AAV-Rec2-CasRx-sgK2 treatment are potential strategies for OP treatment.