Improving oral bioavailability of cyclic peptides by N-methylation

The renaissance of peptides in pharmaceutical industry results from their importance in many biological functions. However, low metabolic stability and the lack of oral availability of most peptides is a certain limitation. Whereas metabolic instability may be often overcome by development of small cyclic peptides containing d-amino acids, the very low oral availability of most peptides is a serious limitation for some medicinal applications. The situation is complicated because a twofold optimization – biological activity and oral availability – is required to overcome this problem. Moreover, most simple “rules” for achieving oral availability are not general and are applicable only to limited cases. Many structural modifications for increasing biological activities and metabolic stabilities of cyclic peptides have been described, of which N-alkylation is probably the most common. This mini-review focuses on the effects of N-methylation of cyclic peptides in strategies to optimize bioavailabilities.     

Effective cancer therapy based on selective drug delivery into cells across their membrane using receptor-mediated endocytosis

Cancer is one of the major causes of death globally. The current treatment options are insufficient, leading to unmet medical needs in cancer treatment. Off-target side effects, multidrug resistance, selective distribution to cancerous tissues, and cell membrane permeation of anti-cancer agents are critical problems to overcome. There is a method to solve these problems by using receptor-mediated endocytosis (RME). It is well known that proteins such as integrin, HER2, EGFR, or other cancer biomarkers are specifically overexpressed on the surface of target cancer cells. By taking advantage of such specific receptors, payloads can be transported into cells through endocytosis using a conjugate composed of the corresponding ligands connected to the payloads by an appropriate linker. After RME, the payloads released by endosomal escape into the cytoplasm can exhibit the cytotoxic activity against cancer cells. Cell-penetrating peptides (CPPs), tumor-homing peptides (THPs), and monoclonal antibodies (mAbs) are utilized as ligands in this system. Antibody drug conjugates (ADCs) based on RME have already been used to cure cancer. In addition to the canonical conjugate method, nanocarriers for spontaneous accumulation in cancer tissue due to enhanced permeability and retention (EPR) effect are extensively used. In this review, I introduce the possibilities and advantages of drug design and development based on RME for the treatment of cancer.     

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrB_P223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB_P223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB_P223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB_P223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB_P223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.     

Rab1a rescues the toxicity of PRAF3

The PRA1-superfamily member PRAF3 plays pivotal roles in membrane traffic as a GDI displacement factor via physical interaction with a variety of Rab proteins, as well as in the modulation of antioxidant glutathione through its interaction with EAAC1 (SLC1A1). Overproduction of PRAF3 is known to be toxic to the host cells, although the factors capable of cancelling the toxicity remained unknown. We here show that Rab1a can rescue the cytotoxicity caused by PRAF3 possibly by “positively” regulating ER-Golgi trafficking, cancelling the “negative” modulation by PRAF3. Our results illuminate the close physiological relationship between PRAF3 and Rab proteins.     

Ca2+ signals triggered by bacterial pathogens and microdomains

Recent reports have highlighted the pivotal role of Ca²⁺ during host cell infection by bacterial pathogens. Here, we review how bacterial pore-forming toxins (PFTs) trigger global Ca²⁺ signals to regulate cell adhesion-, inflammatory- or death processes. We comment recent reports describing the role of bacterial effectors injected by a type III secretion system (T3SS) as well as host cell players in the formation of Ca²⁺ microdomains during Shigella invasion and Chlamydia extrusion of host cells. We discuss how modeling and comparison between bacterial-induced and physiological Ca²⁺ microdomains provides insight into the critical parameters shaping the duration of local Ca²⁺ responses.     

C-terminal oligomerization of podocin mediates interallelic interactions

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3′ mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283–382): principally through the first C-terminal helical region (H1, 283–313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332–348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r²?=?0.68, P?=?9.2?×?10^?32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.     

PERK inhibition attenuates the abnormalities of the secretory pathway and the increased apoptotic rate induced by SIL1 knockdown in HeLa cells

Loss-of-function mutations in the SIL1 gene are linked to Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of infancy characterized by cerebellar and skeletal muscle degeneration. SIL1 is a ubiquitous adenine nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone BiP. The complexity of mechanisms by which loss of SIL1 causes MSS is not yet fully understood. We used HeLa cells to test the hypothesis that impaired protein folding in the ER due to loss of SIL1 could affect secretory trafficking, impairing the transport of cargoes essential for the function of MSS vulnerable cells. Immunofluorescence and ultrastructural analysis of SIL1-knocked-down cells detected ER chaperone aggregation, enlargement of the Golgi complex, increased autophagic vacuoles, and mitochondrial swelling. SIL1-interefered cells also had delayed ER-to-plasma membrane transport with retention of Na⁺/K⁺-ATPase and procollagen-I in the ER and Golgi, and increased apoptosis. The PERK pathway of the unfolded protein response was activated in SIL1-interfered cells, and the PERK inhibitor GSK2606414 attenuated the morphological and functional alterations of the secretory pathway, and significantly reduced cell death. These results indicate that loss of SIL1 is associated with alterations of secretory transport, and suggest that inhibiting PERK signalling may alleviate the cellular pathology of SIL1-related MSS.     

以谷胱甘肽为电子供体的细胞膜氧化还原系统

内载谷胱甘肽(GSH)的大豆(Glycine max L.)下胚轴正向型质膜囊泡具有以GSH为电子供体的跨膜电子传递活性,能还原膜外电子受体FeCN和细胞色素(Cyt)C,其还原速率分别为(21.6±0.6)nmolFeCN·min~(-1)·mg~(-1)蛋白和(6.6±1.0)nmol Cyt C·min~(-1)·mg~(-1)蛋白。这种跨膜电子传递能引起膜上Cyt P-450吸收光谱标志带(Soret带)的变化,表明Cyt P-450参与了这一氧化还原过程。在跨质膜电子传递的同时伴随着H~ 运输和膜电位的改变。