Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

SUMMARY Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.

Summary In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID-targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss implications of these findings for harnessing antibody diversification mechanisms.

Less than a year since the start of the COVID-19 pandemic, ten vaccines against SARS-CoV-2 have been approved for at least limited use, with over sixty others in clinical trials. This swift achievement has generated excitement and arrives at a time of great need, as the number of COVID-19 cases worldwide continues to rapidly increase. Two vaccines are currently approved for full use, both built on mRNA and lipid nanotechnology platforms, a success story of mRNA technology 20 years in the making. For patients with cancer, questions arise around the safety and efficacy of these vaccines in the setting of immune alterations engendered by their malignancy and/or therapies. We summarize the current data on leading COVID-19 vaccine candidates and vaccination of patients undergoing immunomodulatory cancer treatments. Most current cancer therapeutics should not prevent the generation of protective immunity. We call for more research in this area and recommend that the majority of patients with cancer receive COVID vaccinations when possible.

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The beta-site APP cleaving enzyme-1 (BACE1) mediates the first cleavage of the beta-amyloid precursor protein (APP) to yield the amyloid beta-peptide (Abeta), a key pathogenic agent in Alzheimer's disease (AD). Using a proteomic approach based on in-cell chemical cross-linking and tandem affinity purification (TAP), we herein identify sorting nexin 6 (SNX6) as a BACE1-associated protein. SNX6, a PX domain protein, is a putative component of retromer, a multiprotein cargo complex that mediates the retrograde trafficking of the cation-independent mannose-6-phosphate receptor (CI-MPR) and sortilin. RNA interference suppression of SNX6 increased BACE1-dependent secretion of soluble APP (sAPPbeta) and cell-associated fragments (C99), resulting in increased Abeta secretion. Furthermore, SNX6 reduction led to elevated steady-state BACE1 levels as well as increased retrograde transport of BACE1 in the endocytic pathway, suggesting that SNX6 modulates the retrograde trafficking and basal levels of BACE1, thereby regulating BACE1-mediated APP processing and Abeta biogenesis. Our study identifies a novel cellular pathway by which SNX6 negatively modulates BACE1-mediated cleavage of APP.

A femtosecond‐pulsed Ti:sapphire laser is used to generate surface features in slabs of poly(dimethylsiloxane) with minimum dimensions of 1 μm—smaller than those available by rapid‐prototyping techniques using transparency masks. The fabrication of magnetic field concentrators and the addition of custom features to a generic microfluidic channel (see Figure) demonstrate the utility of the technique.

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1-infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms.