The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAbs ontogeny and mechanisms of binding PfCSP to neutralize sporozoites will facilitate vaccine development. Here, we isolated mAbs clonally related to L9 and showed that this B-cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9k) with clonally-related heavy chains resulted in chimeric mAbs that cross-linked two NVDP, cross-reacted with NANP, and more potently neutralized sporozoites compared to their original light chain. Structural analyses revealed that chimeric mAbs bound the minor repeat motif in a type-1 beta-turn seen in other repeat-specific antibodies. These data highlight the importance of L9k in binding NVDP on PfCSP to neutralize SPZ and suggest that PfCSP-based immunogens might be improved by presenting 2 or more NVDP.

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation

Molecular chaperones, including Hsp70/Hsp40 families, play central roles in binding substrates to prevent their aggregation. How Hsp40s select different conformations of substrates remains poorly understood. Here, we report a novel interaction between the Hsp40 DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a beta-turn element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau beta-turn fragments, but not mutant fragments, can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. This identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3′-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Adaptation of the Romanomermis culicivorax CCA-adding enzyme to miniaturized armless tRNA substrates

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that – when inserted into the human enzyme - confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3’-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Beta Turn Propensity and Polymer Scaling Exponent Identify Intrinsically Disordered Proteins that Phase Separate

The complex cellular milieu can spontaneously de-mix in a process driven in part by proteins that are intrinsically disordered (ID). We hypothesized that protein self-interactions that determine the polymer scaling exponent, v, of monomeric ID proteins (IDPs), also facilitate de-mixing transitions into phase separated assemblies. We analyzed a protein database containing subsets that are folded, ID, or IDPs identified previously to spontaneously phase separate. We found that the subsets differentiated into distinct protein classes according to sequence-based calculations of v and, surprisingly, the propensity in the sequence for adopting the β-turn. Structure-based simulations find that transient β-turn structures reduce the desolvation penalty of forming a protein-rich phase. By this mechanism, β-turns act as energetically favored nucleation points, which may explain the increased propensity for turns in IDPs that are utilized biologically for phase separation.

Insight into the Mechanism of Internalization of the Cell-Penetrating Carrier Peptide Pep-1 by Conformational Analysis

Different secondary structures of the pep-1 protein were blamed for transmembrane internalization process of drugs and drug deliveries. But which structure will be important for transmembrane delivery was still not clear. In this study, interactions between pep-1 and cell membranes were studied. Pep-1 in the buffer (Pep-1) and pep-1 on graphene (PDS/G) or they on graphene oxide (PDS/GO) were composed as the transmembrane delivery system to study the different secondary structure of pep-1 that influence for their transmembrane delivery. The curves of chirascan circular dichroism (CD) and all-atom discontinuous molecular dynamics (DMD) simulations illuminate that, in a buffer environment, most pep-1 formed 3–10 helix structures. Meanwhile, when Pep-1 composed graphene slice and formed PDS/G, 3–10 helix and alpha-helix structures can be found in small quantities. When they on graphene oxide and formed PDS/GO, coil or type II beta-turn structure can be found from most of the pep-1 and 3–10 helix structure disappeared. By using sum-frequency generation (SFG) vibrational spectroscopy, we found that pep-1 with 3–10 helix structures in buffer solutions damaged the lipid bilayer violently. PDS/G with less 3–10 helix structures will change the orientation of lipid bilayer effectively but slightly. Pep-1 with coil or type II Beta-turn in PDS/GO cannot influence the structure of lipid bilayers. Hemolysis experiments also proved that when pep-1 composed as PDS/G, they will change the orientation of the plasma membrane of red blood cells effectively but slightly. When they attach on the GO and formed PDS/GO, the plasma membrane of red blood cells cannot be influenced. In conclusion, 3–10 helix structures will be positively correlated with disturbance of membranes. These results will be effectively guided the clinic application of pep-1 as a transporter of the drug delivery system.

LFB: A Novel Antimicrobial Brevinin-Like Peptide from the Skin Secretion of the Fujian Large Headed Frog, Limnonectes fujianensi

Amphibians are a natural source of abundant antimicrobial peptides and thus have been widely investigated for isolation of such biomolecules. Many new antimicrobial peptide families have been discovered from amphibians. In this study, a novel antimicrobial peptide named Limnonectes fujianensis Brevinvin (LFB) has been identified in the skin secretion from the Fujian large headed frog, Limnonectes fujianensis. The cDNA sequence was cloned from a skin secretion library and the predicted mature peptide was identified through MS/MS fragmentation sequencing of reverse phase HPLC fractions on the same sample. LFB was predicted to be an amphipathic, hydrophobic, alpha helical, and beta turn peptide that inserts into a lipid bilayer in order to kill the cells. In antimicrobial assays, a synthetic replicate of this novel antimicrobial peptide demonstrated significant activity against the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast, Candida albicans. This novel peptide was highly potent (MIC 4.88 uM) against Gram-negative bacterium, and also has the ability to inhibit the growth of human cancer cell lines with IC50 values ranging from 18.9 μM down to 2.0 μM. These findings help to enrich our understanding of Brevinin-like peptides. Moreover, the data presented here validate frog secretion as a source of potential novel antimicrobial peptides, that also exhibit anti-tumor properties, that could be useful for the treatment of cancer.

Novel Technologies for Dipeptide Drugs Design and their Implantation

The article is an overview of author’s data obtained in the framework of the project “The Creation of dipeptide preparations” at the V.V. Zakusov Institute of Pharmacology, Moscow, Russia. Advantages of dipeptides over longer peptides consist in that they are orally active owing to higher stability and ability to penetrate biological barriers due to the presence of specific ATP–dependent transporters in enterocytes and blood-brain barrier. Two original approaches for dipeptide drugs design have been developed. Both of them are based on the idea of a leading role of central dipeptide fragment of the peptide chain beta-turn in the peptide-receptor interaction. The first approach, named "peptide drug-based design" represents the transformation of known nonpeptide drug into its dipeptide topological analog. The latter usually corresponds to a beta-turn of some regulatory peptide. The second approach represents the design of tripeptoide mimetic of the beta-turn of regulatory peptide or protein. The results of the studies, which led to the discovery of endogenous prototypes of the known non-peptide drugs piracetam and sulpiride, are presented herein. The paper discusses the process, based on the abovementioned principles, that was used in designing of nontoxic, orally available, highly effective dipeptide drugs: nootropic noopept, dipeptide analog of piracetam; antipsychotic dilept, neurotensin tripeptoid analog; selective anxiolytic GB-115, tripeptoid analog of CCK-4, and potential neuroprotector GK-2, homodimeric dipeptide analog of NGF.