C. Stephens

University of California, San Francisco

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Judith A. Clements

Translational Research Institute

Scott H. Stansfield

Monash Health

Niek van Hilten

University of California, San Francisco

Lisa Zheng

University of California, San Francisco

Michael Grabe

University of California, San Francisco

P. V. Danenberg

University of Southern California

Philip C. Mack

Icahn School of Medicine at Mount Sinai

K. D. Danenberg

University Hospital Carl Gustav Carus

David R. Gandara

University of California, Davis

Daniela Loessner

Monash University

Cooperative Institutions

Queensland University of Technology

AstraZeneca (United Kingdom)

Translational Research Institute

University of Southern California

Princess Alexandra Hospital

University of Cologne

AstraZeneca (Brazil)

University of Queensland

University of Tennessee at Knoxville

The University of Texas MD Anderson Cancer Center

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Robust lipid scrambling by TMEM16 proteins requires an open groove and thin membrane

bioRxiv (Cold Spring Harbor Laboratory) (2024)

C. Stephens Niek van Hilten Lisa Zheng Michael Grabe

Biological membranes are complex and dynamic structures with different populations of lipids on their inner and outer leaflets. The Ca<2+/>-activated TMEM16 family of membrane proteins play an important role in collapsing this asymmetric lipid distribution by spontaneously, and bidirectionally, scrambling phospholipids between the two leaflets, which can initiate signaling and alter the physical properties of the membrane. While evidence shows that lipid scrambling occurs via an open hydrophilic pathway ("groove") that spans the membrane, it remains unclear if all family members facilitate lipid movement in this manner. Here we present a comprehensive computational study of lipid scrambling by all TMEM16 members with experimentally solved structures. We performed coarse-grained molecular dynamics (MD) simulations of 27 structures from five different family members solved under activating and non-activating conditions, and we captured over 700 scrambling events in aggregate. This enabled us to directly compare scrambling rates, mechanisms, and protein-lipid interactions for fungal and mammalian TMEM16s, in both open (Ca<2+/>-bound) and closed (Ca<2+/>-free) conformations with statistical rigor. We show that nearly all (>90%) scrambling occurs in the dilated groove and the degree of induced membrane thinning positively correlates with the scrambling rate. Surprisingly, we also observed 60 scrambling events that occurred outside the canonical groove, over 90% of which took place at the dimer-dimer interface in mammalian TMEM16s. This new site suggests an alternative mechanism for lipid scrambling in the absence of an open groove.

Scrambling

Phospholipid scramblase

10.1101/2024.09.25.615027

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523: Identification of kallikrein-related peptidase 7 degradome and transcriptome targets in ovarian cancer

European Journal of Cancer (2014)

L. Munasinghage Silva Thomas Stoll Scott H. Stansfield C. Stephens Marcus L. Hastie

Identification

10.1016/s0959-8049(14)50465-0

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[25] Use of PCR in detection of antisense transcripts in HTLV-I-infected patients and human T-cell lines

Methods in neurosciences (1995)

Syamalima Dube Benjamin Z.S. Paul Virginia Bryz‐Gornia C. Stephens Selda Erensoy

Sense strand

Transcription

Primer (cosmetics)

Coding strand

10.1016/s1043-9471(06)80103-5

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Citations (4)

251 Kallikrein-related Peptidase 4 and Prostate Cancer – an Extended Role in Tumour Progression

European Journal of Cancer (2012)

Ruth A. Fuhrman‐Luck Scott H. Stansfield C. Stephens Daniela Loessner Judith A. Clements

10.1016/s0959-8049(12)70946-2

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Simulation-based survey of TMEM16 family reveals that robust lipid scrambling requires an open groove

C. Stephens Niek van Hilten Lisa Zheng Michael Grabe

Biological membranes are complex and dynamic structures with different populations of lipids in their inner and outer leaflets. The Ca 2+ -activated TMEM16 family of membrane proteins plays an important role in collapsing this asymmetric lipid distribution by spontaneously, and bidirectionally, scrambling phospholipids between the two leaflets, which can initiate signaling and alter the physical properties of the membrane. While evidence shows that lipid scrambling can occur via an open hydrophilic pathway (“groove”) that spans the membrane, it remains unclear if all family members facilitate lipid movement in this manner. Here we present a comprehensive computational study of lipid scrambling by all TMEM16 members with experimentally solved structures. We performed coarse-grained molecular dynamics (MD) simulations of 27 structures from five different family members solved under activating and non-activating conditions, and we captured over 700 scrambling events in aggregate. This enabled us to directly compare scrambling rates, mechanisms, and protein-lipid interactions for fungal and mammalian TMEM16s, in both open (Ca 2+ -bound) and closed (Ca 2+ -free) conformations with statistical rigor. We show that all TMEM16 structures thin the membrane and that the majority of (>90%) scrambling occurs at the groove only when TM4 and TM6 have sufficiently separated. Surprisingly, we also observed 60 scrambling events that occurred outside the canonical groove, over 90% of which took place at the dimer-dimer interface in mammalian TMEM16s. This new site suggests an alternative mechanism for lipid scrambling in the absence of an open groove.

Scrambling

Groove (engineering)

10.7554/elife.105111.1

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Author response: Simulation-based survey of TMEM16 family reveals that robust lipid scrambling requires an open groove

C. Stephens Niek van Hilten Lisa Zheng Michael Grabe

Scrambling

10.7554/elife.105111.1.sa4

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009. KALLIKREIN-RELATED PROTEASES AS NOVEL THERAPEUTIC TARGETS IN PROSTATE AND OVARIAN CANCER

Reproduction Fertility and Development (2009)

Judith A. Clements Yang Dong Daniela Loessner Olivia L. Tan Shirly Sieh

The kallikrein-related (KLKs) peptidases are implicated in prostate and ovarian cancer invasion/metastasis via activation of growth factors, proteases and extracellular matrix degradation involved in. In our published work, we used cell biology approaches to show novel associations of KLK peptidases with processes indicative of metastasis and the potential of our novel sunflower trypsin inhibitor scaffold-engineered KLK4 inhibitor. Our current studies are directed towards discovering the precise KLK target proteins/substrates and the subsequent signalling pathways involved in these events in order to determine their therapeutic target potential. In this regard, we are using novel tissue engineered biomimetic 3D gel matrices to better mimic the in vivo micro-environment of prostate cancer cells especially in bone metastasis and peritoneal invasion in ovarian cancer. Pilot studies show that PC3 cells cultured on an osteoblast-derived bone matrix undergo an EMT-like change but remain dispersed on the cell surface. In contrast, LNCaP cells cluster aligning with the fibrillar structure as they invade into the bone matrix as typically seen in vivo. KLK4 proteolysis of the osteoblast-derived bone matrix has identified additional novel substrates. In addition, we are exploring the cell biology that underlies the reported high KLK4 or KLK7 levels associated with poorer outcome in women with epithelial ovarian cancer (EOC). Of note, KLK4 or KLK7 transfected SKOV3 EOC cells have increased chemoresistance to taxol and/or cisplastin suggesting a mechanism for this poor outcome. Furthermore, KLK7 transfected SKOV-3 cells form multicellular aggregates (MCA) in agarose suspension (a process indicative of peritoneal tumour cell spread seen in ascites fluid clinically) which can be reversed by a KLK7 blocking antibody indicating the critical role played by KLK7 in this event. These new paradigms are providing novel information on the role of KLK peptidases in prostate and ovarian cancer progression and their potential as novel therapeutic targets.

10.1071/srb09abs009

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Phase I clinical and translational evaluation of AZD6738 in combination with durvalumab in patients (pts) with lung or head and neck carcinoma

Annals of Oncology (2018)

Matthew Krebs Juanita Lopez Anthony B. El-Khoueiry Yung‐Jue Bang Sophie Postel‐Vinay

Durvalumab

Tolerability

10.1093/annonc/mdy279.401

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Citations (7)

142: Proteolytic regulation of the EphB4 receptor in prostate cancer

European Journal of Cancer (2014)

Jessica Lisle Inga Mertens‐Walker C. Stephens Scott H. Stansfield Adrian C. Herington

10.1016/s0959-8049(14)50120-7

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A novel RT-PCR approach to detecting EML4-ALK fusion genes in archival NSCLC tissue.

Journal of Clinical Oncology (2010)

P. V. Danenberg C. Stephens J. Cooc David R. Gandara Philip C. Mack

10535 Background: The echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) fusion gene can be detected in subsets of NSCLC, especially never-smoking patients with adenocarcinoma, and appears to be mutually exclusive of EGFR mutation. PF-1066, a potent and selective c-MET/ALK inhibitor, yields high response rates in ALK-positive patients, as detected by labor-intensive fluorescent in situ hybridization (FISH) methodology. Existing RT-PCR assays for these gene variants are designed to amplify large cDNA fragments (> 450 bases), while formalin-fixed paraffin-embedded (FFPE) specimens yield mostly RNA fragments of < 150 bases. Thus, our goal was to design a robust RT-PCR assay for EML4-ALK fusion gene transcripts suitable for use with commonly available FFPE tissues of limited size. Methods: Synthetic fragments representing the nine EML4-ALK fusion genes variants 1, 2, 3a, 3b, 4, 5a, 5b, 6 and 7 were generated by recursive PCR technology. The approach consisted of amplifying over-lapping fragments containing the fusion variant sequences with outside 5′ and 3′ primers. Primer probes were designed to detect specific EML4-ALK fusion gene fragments with a maximum amplicon of 170 bases by RT-PCR. Results: Functional RT-PCR assay were developed for each specific EML4-ALK variant, meeting criteria specified above for optimal clinical testing. Assays detecting multiple fusion variants without distinguishing specific variants were also designed. All nine known EML4-ALK variants can be detected using 6 RT-PCR assays. Screening of NSCLC cDNAs from the Response Genetics database is underway (molecular and clinical correlations to be presented). Conclusions: We developed RT-PCR assays capable of detecting EML4-ALK fusion gene variants from FFPE tissues. Two assays were successfully established: one that can detect and identify each individual variant, and one capable of detecting the presence of any variant as a single assay. We expect this methodology to provide a useful tool for large-scale screening of NSCLC or other FFPE tissues for the EML4-ALK fusion gene. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics Amgen, AstraZeneca, Biodesix, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, GlaxoSmithKline, Lilly, Merck, Novartis, Pfizer, sanofi-aventis Response Genetics DxS Abbott Laboratories, Bristol-Myers Squibb, Genentech, Lilly, Merck, Novartis, Pfizer

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Amplicon

10.1200/jco.2010.28.15_suppl.10535

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Citations (14)