Richard F. Wagner

Mohs micrographic surgery

The term Mohs chemosurgery, which was coined in the 1930s, was used to describe a new technique for the removal of skin cancers. The method achieved a high degree of precision and conservation during skin cancer surgery. The accuracy of Mohs surgery was not based on the guesswork of representative or incomplete margin samples, as in routine scalpel surgery. Accuracy was ensured by a microscopic survey of all margins. This enabled complete removal of the cancer with surgical borders of 1 or 2 mm. Today Mohs surgery is performed in major university medical centers throughout the country and its accessibility has helped set a new standard for quality patient care. While the routine techniques for removing nonmelanoma primary skin malignancies (including cryosurgery, ionizing radiation, curettage, and standard scalpel excision) will remain the accepted practice for the treatment of the majority of skin tumors, Mohs surgery is the treatment of choice for certain cancers.

Single Particle Characterization of Iron-induced Pore-forming α-Synuclein Oligomers

Aggregation of α-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored α-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers (“intermediate I”). Under these conditions, Fe3+ at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers (“intermediate II”). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe3+-induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomer-specific A11 antibody. Moreover, baicalein and N′-benzylidene-benzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited α-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

IFNα induces Fas expression and apoptosis in hedgehog pathway activated BCC cells through inhibiting Ras-Erk signaling

Basal cell carcinoma (BCC), the most common form of human cancer, is understood to be associated with activation of the sonic hedgehog pathway, through loss-of-function mutations of tumor suppressor PTCH1 or gain-of-function mutations of smoothened. Interferon (IFN)-based therapy is quite effective in BCC treatment, but the molecular basis is not well understood. Here we report a novel mechanism by which IFNα mediates apoptosis in BCCs. In the presence of IFNα, we observed increased apoptosis in a BCC cell line ASZ001, in which PTC is null, and therefore with constitutive activation of the sonic hedgehog pathway. We demonstrate that SMO agonist Ag-1.4 mediates activation of extracellular signal-regulated kinase (Erk) phosphorylation, which is abrogated by IFNα in sonic hedgehog responsive C3H10T1/2 cells. In transient transfection experiments, we demonstrate that IFNα inhibits Erk phosphorylation and serum response element activation induced by expression of SMO, Gli1, PDGFRα and activated Raf, but not activated mitogen-activated Erk-regulating kinase (Mek), suggesting that IFNα targets mainly on Mek function. We further show that IFNα induces expression of Fas in BCC cells through interfering with Mek function. The role of the Fas-L/Fas signaling axis in IFNα-mediated apoptosis is demonstrated by the fact that addition of Fas-L neutralizing antibodies, just as caspase-8 inhibitor Z-IETD-FMK, effectively prevents IFNα-mediated apoptosis. Thus, our data indicate that IFNα-based BCC therapy induces Fas expression and apoptosis through interfering with Mek function.

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein‐conducting channel, the Sec61 complex. The presence of large, water‐filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)‐binding motif in the cytosolic N‐terminus of mammalian Sec61α that bound CaM but not Ca2+‐free apocalmodulin with nanomolar affinity and sequence specificity. In single‐channel measurements, CaM potently mediated Sec61‐channel closure in Ca2+‐dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+‐CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

The chloroplast outer envelope protein OEP37 is a member of the growing β-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance (λ) of Λ= 500 picosiemens (symmetrical 250 mm KCl). The OEP37 channel is cation-selective (PK+/PK– = 14:1) with a voltage-dependent open probability maximal at Vmem = 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d = 3.0 nm and the restriction zone by d = 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.

Sec61 complexes form ubiquitous ER Ca2+ leak channels

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis and in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous pathway for transporting newly synthesized polypeptides into the ER lumen and may also allow calcium leakage from the ER into the cytosol. In this study, planar lipid bilayer experiments demonstrated that the Sec61 complex is permeable to calcium ions. We also investigated whether silencing the SEC61A1 gene affected calcium leakage from the ER. Silencing the SEC61A1 gene using two different siRNAs in HeLa cells for 96 hours had little effect on cell growth and viability. However, calcium leakage from the ER was greatly decreased in the SEC61A1-silenced cells. Thus, the Sec61 complexes that are present in the ER membrane of nucleated cells form calcium leak channels that may play a crucial role in calcium homeostasis.

The Mitochondrial Oxidase Assembly Protein1 (Oxa1) Insertase Forms a Membrane Pore in Lipid Bilayers

Background: Oxa1 mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. Results: Oxa1 forms a voltage- and substrate-dependent membrane pore. Conclusion: The channel properties of the Oxa1 pore are compatible with the membrane-potential regulated protein insertase. Significance: This is the first report on the pore-forming capacity of Oxa1, providing mechanistic insight into the insertase mechanism of Oxa1. The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.

Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase. NEW PERSPECTIVES FROM COMPARATIVE SINGLE CHANNEL ELECTROPHYSIOLOGY

Background: The archaic translocase of the outer mitochondrial membrane (ATOM) from Trypanosoma brucei mediates protein import. Results: ATOM forms a hydrophilic transmembrane pore with channel characteristics resembling bacterial-type protein translocases. Conclusion: ATOM descended from a bacterial porin and represents an evolutionary intermediate. Significance: ATOM presumably represents the missing link between the mitochondrial outer membrane protein import pore and its bacterial ancestors. Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes.

The channel-forming Sym1 protein is transported by the TIM23 complex in a presequence-independent manner.

ABSTRACT The majority of multispanning inner mitochondrial membrane proteins utilize internal targeting signals, which direct them to the carrier translocase (TIM22 complex), for their import. MPV17 and its Saccharomyces cerevisiae orthologue Sym1 are multispanning inner membrane proteins of unknown function with an amino-terminal presequence that suggests they may be targeted to the mitochondria. Mutations affecting MPV17 are associated with mitochondrial DNA depletion syndrome (MDDS). Reconstitution of purified Sym1 into planar lipid bilayers and electrophysiological measurements have demonstrated that Sym1 forms a membrane pore. To address the biogenesis of Sym1, which oligomerizes in the inner mitochondrial membrane, we studied its import and assembly pathway. Sym1 forms a transport intermediate at the translocase of the outer membrane (TOM) complex. Surprisingly, Sym1 was not transported into mitochondria by an amino-terminal signal, and in contrast to what has been observed in carrier proteins, Sym1 transport and assembly into the inner membrane were independent of small translocase of mitochondrial inner membrane (TIM) and TIM22 complexes. Instead, Sym1 required the presequence of translocase for its biogenesis. Our analyses have revealed a novel transport mechanism for a polytopic membrane protein in which internal signals direct the precursor into the inner membrane via the TIM23 complex, indicating a presequence-independent function of this translocase.

Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10.

The endoplasmic reticulum–mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.