Introduction Most of the approximately 60 genes that if mutated cause steroid-resistant nephrotic syndrome (SRNS) are highly expressed in the glomerular podocyte, rendering SRNS a “podocytopathy.” Methods We performed whole-exome sequencing (WES) in 1200 nephrotic syndrome (NS) patients. Results We discovered homozygous truncating and homozygous missense mutation in SYNPO2 (synaptopodin-2) (p.Lys1124∗ and p.Ala1134Thr) in 2 patients with childhood-onset NS. We found SYNPO2 expression in both podocytes and mesangial cells; however, notably, immunofluorescence staining of adult human and rat kidney cryosections indicated that SYNPO2 is localized mainly in mesangial cells. Subcellular localization studies reveal that in these cells SYNPO2 partially co-localizes with α-actinin and filamin A−containing F-actin filaments. Upon transfection in mesangial cells or podocytes, EGFP-SYNPO2 co-localized with α-actinin-4, which gene is mutated in autosomal dominant SRNS in humans. SYNPO2 overexpression increases mesangial cell migration rate (MMR), whereas shRNA knockdown reduces MMR. Decreased MMR was rescued by transfection of wild-type mouse Synpo2 cDNA but only partially by cDNA representing mutations from the NS patients. The increased mesangial cell migration rate (MMR) by SYNPO2 overexpression was inhibited by ARP complex inhibitor CK666. SYNPO2 shRNA knockdown in podocytes decreased active Rac1, which was rescued by transfection of wild-type SYNPO2 cDNA but not by cDNA representing any of the 2 mutant variants. Conclusion We show that SYNPO2 variants may lead to Rac1-ARP3 dysregulation, and may play a role in the pathogenesis of nephrotic syndrome.
Protein-based encapsulation systems have a wide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces. By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules. Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles. We describe an encapsulation system based on the hepatitis B virus capsid in which an engineered high-affinity interaction between cargo and capsid proteins can be modulated by Ca . Cargo proteins are loaded into capsids in the presence of Ca , while ligand removal triggers unbinding inside the container. We observe that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with β-glucosidase activity.
Protein-based encapsulation systems have aw ide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces.By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules.Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles.W ed escribe an encapsulation system based on the hepatitis Bv irus capsid in which an engineered highaffinity interaction between cargo and capsid proteins can be modulated by Ca 2+ .Cargo proteins are loaded into capsids in the presence of Ca 2+ ,while ligand removal triggers unbinding inside the container.W eo bserve that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with b-glucosidase activity.Protein-based nanocontainers provide tantalizing opportunities for the encapsulation and targeted delivery of various drugs and imaging probes,t he construction of nanoreactors, and the development of biomaterials. [1] Virus-like particles (VLPs) have been widely used as encapsulation systems. [1] In natural container systems,l oading and release of cargo is typically exquisitely controlled. To facilitate an increased control of loading in engineered VLPs,s everal groups have mimicked natural container systems and introduced sites for noncovalent interactions between cargo and capsid proteins.Fore xample,V LPs have been modified to have highly negatively charged interiors to efficiently encapsulate cargo proteins tagged with positively charged sequence. [2] Specific protein-protein interactions have also been used to drive encapsulation selectivity by adding complementary coiledcoil sequences to capsid and guest proteins. [3] It has also been possible to use native RNA-capsid interactions to drive encapsulation of cargo into virus-derived VLPs. [4] An approach for metal-induced assembly and encapsulation has also been described. [5] An additional level of control might be achieved if the interaction between cargo and capsid proteins can be modulated post-encapsulation. In many applications,it would be highly beneficial to be able to turn off the noncovalent interaction between cargo and capsid after encapsulation. In protein delivery applications,t he cargo would ideally be separated from capsomers once capsids have disassembled. In nanoreactors,d etachment of enzymes attached to the container wall to move freely in the lumen of the capsid could enable more efficient catalysis and facilitate noncovalent interactions between proteins involved in multienzyme reaction cascades.Freely diffusing proteins inside the nanocontainer would also facilitate the study of protein crowding and protein-protein...
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