Surface Characterization of Insulin Protofilaments and Fibril Polymorphs Using Tip-Enhanced Raman Spectroscopy (TERS)

Amyloid fibrils are β-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimer’s and Parkinson’s diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs.     

Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

Desmosomal cadherins are transmembrane adhesion molecules that provide cell adhesion by interacting in the intercellular space of adjacent cells. In keratinocytes, several desmoglein (Dsg1–4) and desmocollin (Dsc1–3) isoforms are coexpressed. We have shown previously that Dsg2 is less important for keratinocyte cohesion compared with Dsg3 and that the latter forms a complex with p38 MAPK. In this study, we compared the involvement of Dsg2 and Dsg3 in the p38 MAPK-dependent regulation of keratinocyte cohesion. We show that loss of cell adhesion and keratin filament retraction induced by Dsg3 depletion is ameliorated by specific p38 MAPK inhibition. Furthermore, in contrast to depletion of Dsg2, siRNA-mediated silencing of Dsg3 induced p38 MAPK activation, which is in line with immunoprecipitation experiments demonstrating the interaction of activated p38 MAPK with Dsg3 but not with Dsg2. Cell fractionation into a cytoskeleton-unbound and a cytoskeleton-anchored desmosome-containing pool revealed that Dsg3, in contrast to Dsg2, is present in relevant amounts in the unbound pool in which activated p38 MAPK is predominantly detectable. Moreover, because loss of cell adhesion by Dsg3 depletion was partially rescued by p38 MAPK inhibition, we conclude that, besides its function as an adhesion molecule, Dsg3 is strengthening cell cohesion via modulation of p38 MAPK-dependent keratin filament reorganization. Nevertheless, because subsequent targeting of Dsg3 in Dsg2-depleted cells led to drastically enhanced keratinocyte dissociation and Dsg2 was enhanced at the membrane in Dsg3 knockout cells, we conclude that Dsg2 compensates for Dsg3 loss of function.     

Challenges and Opportunities in Radiation-induced Hemorrhagic Cystitis

As diagnosis and treatment of cancer is improving, medical and social issues related to cancer survivorship are becoming more prevalent. Hemorrhagic cystitis (HC), a rare but serious disease that may affect patients after pelvic radiation or systemic chemotherapy, has significant unmet medical needs. Although no definitive treatment is currently available, various interventions are employed for HC. Effects of nonsurgical treatments for HC are of modest success and studies aiming to control radiation-induced bladder symptoms are lacking. In this review, we present current and advanced therapeutic strategies for HC to help cancer survivors deal with long-term urologic health issues.     

A Bacterial Electron-bifurcating Hydrogenase

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.