Publications by authors named "W R NICKEL"

165 Publications

Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation.

Anal Chem 2020 11 14;92(22):14861-14866. Epub 2020 Oct 14.

Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69 120 Heidelberg, Germany.

In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure-function relationship of membrane-inserted oligomers.
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http://dx.doi.org/10.1021/acs.analchem.0c03276DOI Listing
November 2020

FGF2 and IL-1β - explorers of unconventional secretory pathways at a glance.

J Cell Sci 2020 11 5;133(21). Epub 2020 Nov 5.

Heidelberg University Biochemistry Center, Heidelberg 69120, Germany

Fibroblast growth factor 2 (FGF2) and interleukin 1β (IL-1β) were among the earliest examples of a subclass of proteins with extracellular functions that were found to lack N-terminal secretory signal peptides and were shown to be secreted in an ER- and Golgi-independent manner. Many years later, a number of alternative secretory pathways have been discovered, processes collectively termed unconventional protein secretion (UPS). In the course of these studies, unconventional secretion of FGF2 and IL-1β were found to be based upon distinct pathways, mechanisms and molecular machineries. Following a concise introduction into various pathways mediating unconventional secretion and transcellular spreading of proteins, this Cell Science at a Glance poster article aims at a focused analysis of recent key discoveries providing unprecedented detail about the molecular mechanisms and machineries driving FGF2 and IL-1β secretion. These findings are also highly relevant for other unconventionally secreted cargoes that, like FGF2 and IL1β, exert fundamental biological functions in biomedically relevant processes, such as tumor-induced angiogenesis and inflammation.
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http://dx.doi.org/10.1242/jcs.250449DOI Listing
November 2020

Risk Factors for Failure of Nonoperative Treatment of Posterior Shoulder Labral Tears on Magnetic Resonance Imaging.

Mil Med 2020 09;185(9-10):e1556-e1561

Department of Orthopaedic Surgery United States Naval Academy, Naval Health Clinic Annapolis, 121 Blake Rd, Annapolis, MD 21402.

Introduction: There are no reports in the literature describing risk factors for failure of nonoperative treatment of patients with posterior labral tears on magnetic resonance imaging (MRI). The purpose of this study is to identify risk factors for failure of nonoperative treatment in patients with an isolated posterior glenoid labral tear identified on MRI only. Patients with posterior labral tears on MRI who fail to improve with nonoperative treatment likely share a constellation of clinical history, physical exam, and radiographic findings.

Methods: One hundred and fifty-nine active duty military service members under the age of 40 with a posterior labral tear seen on MRI and who were clinically evaluated by a musculoskeletal trained physician were identified. We retrospectively evaluated their records ensuring a minimum of 2 years follow-up after MRI to identify surgical intervention for the posterior labral tear during this time period. Patients were stratified into two groups, those treated with any combination of nonoperative modalities and those treated with posterior labral repair surgery during the 2 years after the MRI. The electronic medical records were reviewed for clinical presentation and physical exam results. We measured multiple radiographic parameters, including glenoid version, size of the tear, and bone loss on MRI. Qualitative and quantitative data were compared between groups using Fisher's exact test and Student's t-test, respectively. This study was conducted under institutional review board approval.

Results: Of the 157 patients' shoulders in our study, 52% (n = 82) of patients with posterior labral tears underwent nonoperative treatment while 48% (n = 75) underwent surgery. The significant risk factors associated with surgery were a history of a specific injury, primary presenting complaint of instability, patient reported history of subluxation, inability to trust their shoulder with overhead activity, decreased strength with weight lifting, positive posterior load/shift exam, positive anterior apprehension, increased osseous glenoid retroversion, increased humeral head subluxation ratio, and anterior labral height (P < 0.05). Patients with a chief complaint of pain were much more likely to succeed with nonoperative treatment while those with instability underwent surgery more often. Ten (12.5%) of the surgical procedures included an anterior and posterior labral repair/stabilization procedure.

Conclusion: Patients with an MRI confirmed posterior labral tear, which present with subjective complaints and physical exam maneuvers consistent with instability, appear less likely to be treated nonoperatively. Increased glenoid retroversion and posterior humeral head subluxation may also predispose patients toward surgical treatment. Additionally, posterior labral tears may extend into the anterior labrum more frequently than is recognized on MRI.
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http://dx.doi.org/10.1093/milmed/usaa122DOI Listing
September 2020

The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P facilitating unconventional secretion of Fibroblast Growth Factor 2.

Commun Biol 2020 03 25;3(1):141. Epub 2020 Mar 25.

Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120, Heidelberg, Germany.

FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces.
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http://dx.doi.org/10.1038/s42003-020-0871-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096399PMC
March 2020

Function of the Medical Team Quarterback: Patient, Family, and Physician Perspectives on Team Care Coordination in Patient- and Family-Centered Primary Care.

Perm J 2019 26;23. Epub 2019 Aug 26.

Kaiser Permanente Washington Health Research Institute, Seattle, WA.

Context: Patient- and family-centered care (PFCC) literature is growing, but few reports present patient, caregiver, and practitioner perspectives about care coordination in a team-based model.

Objective: To understand the patient's, caregiver's, and physician's ideal forms of PFCC, we investigated the function of the medical team quarterback, who coordinates and advocates for appropriate care, and probed to understand how the quarterback works with a team to contribute to ideal PFCC.

Design And Main Outcome Measures: Nine focus groups with 92 participants were held in 3 major cities. Patients (n = 35) and family members (n = 36) were recruited through market research groups. Physicians (n = 21) were recruited by the American College of Physicians. Focus group transcripts were analyzed and coded using inductive analysis.

Results: The quarterback emerged as an important function for addressing care gaps and improving the care experience. We identified 6 themes articulated by participants that defined the role of a medical team quarterback: Overseeing care; coordinating diagnoses, tests, and treatments; advocating for patients; identifying and respecting patient values; proactively communicating; and solving problems. Patients and family members in our sample were open to different members of the care team acting as quarterback in coordination with the physician.

Conclusion: Medical team quarterbacks were perceived as enhancing team-based care by facilitating the coordination/communication that is critical to PFCC. Patients and family members acknowledged that PFCC can be delivered by different members of the medical team if the care felt organized and coordinated with the primary care physician.
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http://dx.doi.org/10.7812/TPP/18.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730945PMC
February 2020

A time-resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling.

FEBS Lett 2019 08 7;593(16):2162-2176. Epub 2019 Jun 7.

Heidelberg University Biochemistry Center, Germany.

Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induced angiogenesis. Here, we describe a novel time-resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose-response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2-dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.
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http://dx.doi.org/10.1002/1873-3468.13462DOI Listing
August 2019

U.S. Internists' Perspectives on Discussing Cost of Care With Patients: Structured Interviews and a Survey.

Ann Intern Med 2019 05;170(9_Suppl):S39-S45

American College of Physicians, Philadelphia, Pennsylvania (A.W., S.R., C.D.S., W.N.).

Background: Rising out-of-pocket costs are creating a need for cost conversations between patients and physicians.

Objective: To understand the factors that influence physicians to discuss and consider cost during a patient encounter.

Design: Mixed-methods study using semistructured interviews and a survey.

Setting: United States.

Participants: 20 internal medicine physicians were interviewed; 621 internal medicine physician members of the American College of Physicians completed the survey.

Measurements: Interviews were analyzed by using thematic analysis, and surveys were analyzed by using descriptive statistics.

Results: From the interviews, 4 themes were identified: Physicians are 1) aware that patients are struggling to afford medical care; 2) relying on clues from patients that hint at their cost sensitivity; 3) relying on experience to anticipate potentially high-cost treatments; and 4) aware that patients are making financial trade-offs to afford their care. Three quarters (n = 466) of survey respondents stated that they consider out-of-pocket costs when making most clinical decisions. For 31% (n = 191) of participants, there were times in the past year that they wanted to discuss out-of-pocket prescription drug costs with patients but did not. The most influential factors for ordering a test are the desire to be as thorough as possible (71% [n = 422]) and insurance coverage for the test (68% [n = 422]).

Limitation: Findings are self-reported, the sample is limited to a single specialty, the survey response rate was low, information on the patient population was limited, and the survey instrument is not validated.

Conclusion: Physicians are attuned to the burden of health care costs and are willing to consider alternative options based on a patient's cost sensitivity.

Primary Funding Source: Robert Wood Johnson Foundation.
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http://dx.doi.org/10.7326/M18-2136DOI Listing
May 2019

Actions and processes that patients, family members, and physicians associate with patient- and family-centered care.

BMC Fam Pract 2019 02 25;20(1):35. Epub 2019 Feb 25.

TrilliumHealth Partners, 100 Queensway West, 6th, Mississauga, ON, L5B 1B8, Canada.

Background: Patient- and family-centered care (PFCC) is increasingly linked to improved communication, care quality, and patient decision making. However, in order to consistently implement and study PFCC, health care systems and researchers need a solid evidentiary base. Most current definitions and models of PFCC are broad and conceptual, and difficult to translate into measurable behaviors and actions. This paper provides a brief overview of all actions that focus group respondents associated with PFCC in ambulatory (outpatient) care settings and then explores actions associated with the concept of "dignity and respect" in greater detail.

Methods: We conducted nine focus groups with patients, family members, and physicians in three metropolitan regions across the United States. Group discussions were transcribed and analyzed using a thematic analysis approach.

Results: We identified 14 domains and 47 specific actions that patients, family members, and physicians associate with PFCC. In addition to providing a detailed matrix of these domains and actions, this paper details the actions associated with the "dignity and respect" concept. Key domains identified under "dignity and respect" include: 1) building relationships, 2) providing individualized care, and 3) respecting patients' time. Within these domains we identified specific actions that break down these abstract ideas into explicit and measurable units such as taking time, listening, including family, and minimizing wait times. We identified 9, 6, and 3 specific actions associated, respectively, with building relationships, providing individualized care, and respecting patients' time.

Conclusions: Our work fills a critical gap in our ability to understand and measure PFCC in ambulatory care settings by breaking down abstract concepts about PFCC into specific measurable actions. Our findings can be used to support research on how PFCC affects clinical outcomes and develop innovative tools and policies to support PFCC.
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http://dx.doi.org/10.1186/s12875-019-0918-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388493PMC
February 2019

Principles for Patient and Family Partnership in Care: An American College of Physicians Position Paper.

Ann Intern Med 2018 12 27;169(11):796-799. Epub 2018 Nov 27.

David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California (P.G.).

In this position paper, the American College of Physicians (ACP) examines the rationale for patient and family partnership in care and reviews outcomes associated with this concept, including greater adherence to care plans, improved satisfaction, and lower costs. The paper also explores and acknowledges challenges associated with implementing patient- and family-centered models of care. On the basis of a comprehensive literature review and a multistakeholder vetting process, the ACP's Patient Partnership in Healthcare Committee developed a set of principles that form the foundation for authentic patient and family partnership in care. The principles position patients in their rightful place at the center of care while acknowledging the importance of partnership between the care team and patient in improving health care and reducing harm. The principles state that patients and families should be treated with dignity and respect, be active partners in all aspects of their care, contribute to the development and improvement of health care systems, and be partners in the education of health care professionals. This paper also recommends ways to implement these principles in daily practice.
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http://dx.doi.org/10.7326/M18-0018DOI Listing
December 2018

Single event visualization of unconventional secretion of FGF2.

J Cell Biol 2019 02 23;218(2):683-699. Epub 2018 Nov 23.

Heidelberg University Biochemistry Center, Heidelberg, Germany

FGF2 is exported from cells by an unconventional secretory mechanism. Here, we directly visualized individual FGF2 membrane translocation events at the plasma membrane using live cell TIRF microscopy. This process was dependent on both PI(4,5)P-mediated recruitment of FGF2 at the inner leaflet and heparan sulfates capturing FGF2 at the outer plasma membrane leaflet. By simultaneous imaging of both FGF2 membrane recruitment and the appearance of FGF2 at the cell surface, we revealed the kinetics of FGF2 membrane translocation in living cells with an average duration of ∼200 ms. Furthermore, we directly demonstrated FGF2 oligomers at the inner leaflet of living cells with a FGF2 dimer being the most prominent species. We propose this dimer to represent a key intermediate in the formation of higher FGF2 oligomers that form membrane pores and put forward a kinetic model explaining the mechanism by which membrane-inserted FGF2 oligomers serve as dynamic translocation intermediates during unconventional secretion of FGF2.
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http://dx.doi.org/10.1083/jcb.201802008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363455PMC
February 2019

Unconventional Secretion Mediates the Trans-cellular Spreading of Tau.

Cell Rep 2018 05;23(7):2039-2055

Schaller Research Group at the University of Heidelberg and the DKFZ, Proteostasis in Neurodegenerative Disease (B180), INF 581, 69120 Heidelberg, Germany. Electronic address:

The progressive deposition of misfolded hyperphosphorylated tau is a pathological hallmark of tauopathies, including Alzheimer's disease. However, the underlying molecular mechanisms governing the intercellular spreading of tau species remain elusive. Here, we show that full-length soluble tau is unconventionally secreted by direct translocation across the plasma membrane. Increased secretion is favored by tau hyperphosphorylation, which provokes microtubule detachment and increases the availability of free protein inside cells. Using a series of binding assays, we show that free tau interacts with components enriched at the inner leaflet of the plasma membrane, finally leading to its translocation across the plasma membrane mediated by sulfated proteoglycans. We provide further evidence that secreted soluble tau species spread trans-cellularly and are sufficient for the induction of intracellular tau aggregation in adjacent cells. Our study demonstrates the mechanistic details of tau secretion and provides insights into the initiation and progression of tau pathology.
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http://dx.doi.org/10.1016/j.celrep.2018.04.056DOI Listing
May 2018

Unconventional mechanisms of eukaryotic protein secretion.

Curr Biol 2018 04;28(8):R406-R410

Heidelberg University, Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

A big surprise in the molecular cell biology of eukaryotes has been the discovery of pathways of protein secretion that are not linked to the endoplasmic reticulum (ER) and the Golgi apparatus. Various kinds of unconventional secretory processes have been described, including two major pathways for two distinct sets of cargoes that are initially synthesized as soluble proteins in the cytoplasm. These two pathways are mechanistically distinct from one another. One is based upon direct protein translocation across lipidic pores in the plasma membrane (type I unconventional secretion). The second pathway involves the recruitment of cytoplasmic proteins into vesicular compartments of the endocytic membrane system that fuse with the plasma membrane to release proteins into the extracellular space (type III unconventional secretion). This primer highlights the mechanisms and molecular machineries of these pathways that were discovered with fibroblast growth factor 2 (FGF2; type I) and acyl-CoA binding protein (Acb1; type III) as the most prominent cargo proteins. Furthermore, the physiological significance of these secretory routes in both health and disease is discussed for a broader range of cargo proteins.
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http://dx.doi.org/10.1016/j.cub.2017.11.074DOI Listing
April 2018

Unconventional protein secretion: Diversity and consensus.

Semin Cell Dev Biol 2018 11 17;83:1-2. Epub 2018 Mar 17.

Hubrecht Institute of the KNAW & UMC Utrecht, 3584 CT Utrecht, The Netherlands; Department of Cell Biology, UMC Groningen, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.semcdb.2018.03.007DOI Listing
November 2018

A direct gateway into the extracellular space: Unconventional secretion of FGF2 through self-sustained plasma membrane pores.

Semin Cell Dev Biol 2018 11 5;83:3-7. Epub 2018 Mar 5.

Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

As illustrated by a diverse set of examples in this special issue, multiple mechanisms of protein secretion have been identified in eukaryotes that do not involve the endoplasmic reticulum (ER) and the Golgi apparatus. Here we focus on the type I pathway with Fibroblast Growth Factor 2 (FGF2) being the most prominent example. Unconventional secretion of FGF2 from cells is mediated by direct protein translocation across the plasma membrane. A unique feature of this process is the ability of FGF2 to form its own membrane translocation intermediate through oligomerization and membrane insertion. This process depends on the phosphoinositide PI(4,5)P at the inner leaflet and results in the formation of lipidic membrane pores in the plasma membrane. Various lines of evidence suggest that these pores are characterized by a toroidal architecture with FGF2 oligomers being accommodated in the center of these structures. At the outer leaflet of the plasma membrane, membrane proximal heparan sulfate proteoglycans are required for the final step of FGF2 translocation into the extracellular space. Based upon mutually exclusive interactions of FGF2 with PI(4,5)P versus heparan sulfates, an assembly/disassembly pathway has been proposed to be the underlying principle of directional transport of FGF2 across the plasma membrane. Thus, the core mechanism of unconventional secretion of FGF2 is based upon three discrete steps with (i) PI(4,5)P dependent oligomerization of FGF2 at the inner leaflet, (ii) insertion of membrane spanning FGF2 oligomers into the plasma membrane and (iii) disassembly at the outer leaflet mediated by heparan sulfates that subsequently retain FGF2 on cell surfaces. This process has recently been reconstituted with an inside-out membrane model system using giant unilamellar vesicles providing a compelling explanation of how FGF2 reaches the extracellular space in an ER/Golgi independent manner. This review is part of a Special Issue of SCDB on "unconventional protein secretion" edited by Walter Nickel and Catherine Rabouille.
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http://dx.doi.org/10.1016/j.semcdb.2018.02.010DOI Listing
November 2018

Extra-Articular Retained Missiles; Is Surveillance of Lead Levels Needed?

Mil Med 2018 03;183(3-4):e107-e113

Uniformed Services University-Walter Reed Department of Surgery, Walter Reed National Military Medical Center, 8901 Wisconsin Avenue, Bethesda, MD 20889.

Background: Although gunshot wounds are relatively common, lead toxicity associated with extra-articular retained missiles (EARMs) is an uncommon, yet potentially devastating, complication. Although the risk of lead toxicity with intra-articular retained missiles is well known, EARMs are routinely left in situ or only removed in selected circumstances secondary to the relatively rare occurrence of complications.

Methods: We first describe a patient with systemic lead poisoning associated with retained lead fragments after a gunshot-induced left femoral shaft fracture. We then performed a systematic review of the literature to answer the following questions: (1) In the setting of retained extra-articular bullets and/or bullet fragments, is regular monitoring and/or surveillance of lead levels in the blood routinely indicated? and, if so, (2) what are the selected factors that portend an increased risk for elevations in blood lead levels in the setting of retained extra-articular bullets and/or bullet fragments? The systematic review was conducted according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) reporting guidelines, of the English language literature utilizing Medline (PubMed), EMBASE, Cochrane, and CINAHL on the topic of lead poisoning, retained bullet, and gunshot wound, and then searched for additional references by manually searching of bibliographies of the included references. Studies were included if they provided clinical data on one or both of our study questions; included studies were evaluated using the accepted levels of evidence.

Findings: Routine monitoring or surveillance of lead levels in blood is recommended in all cases of EARM at the time of hospital admission and again at discharge, followed by monthly intervals until 3 mo post-injury and then again at 1 yr post-injury. The studies identified demonstrated significant risk factors for elevated blood lead levels in the setting of EARM, which included the number of retained missiles and concomitant fracture.

Discussion: Recommendations for routine monitoring and surveillance of blood lead levels in all cases of EARM are conflicting, but such monitoring appears to be warranted given that the potential risks and morbidity associated with systemic lead poisoning are outweighed by any potential harm of short-term, blood lead level monitoring. Outside of concomitant fracture, the evidence for making further clinical recommendations regarding selected risk factors that portend an increased risk for elevated blood lead levels after gunshot injury is weak. Larger level II and III studies are needed to determine the indications for and frequency of lead toxicity screening after retained EARM.
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http://dx.doi.org/10.1093/milmed/usx076DOI Listing
March 2018

An emerging case for membrane pore formation as a common mechanism for the unconventional secretion of FGF2 and IL-1β.

J Cell Sci 2017 Oct 4;130(19):3197-3202. Epub 2017 Sep 4.

Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.

Extracellular proteins with important signalling roles in processes, such as inflammation and angiogenesis, are known to employ unconventional routes of protein secretion. Although mechanisms of unconventional protein secretion are beginning to emerge, the precise molecular details have remained elusive for the majority of cargo proteins secreted by unconventional means. Recent findings suggest that for two examples of unconventionally secreted proteins, interleukin 1β (IL-1β) and fibroblast growth factor 2 (FGF2), the common molecular principle of pore formation may be shared. Under specific experimental conditions, secretion of IL-1β and FGF2 is triggered by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P]-dependent formation of pores across the plasma membrane. However, the underlying mechanisms are different, with FGF2 known to directly interact with PI(4,5)P, whereas in the case of IL-1β secretion, it is proposed that the N-terminal fragment of gasdermin D interacts with PI(4,5)P to form the pore. Thus, although implemented in different ways, these findings suggest that pore formation may be shared by the unconventional secretion mechanisms for FGF2 and IL-1β in at least some cases. In this Opinion article, we discuss the unconventional mechanisms of FGF2 and IL-1β release with a particular emphasis on recent discoveries suggesting the importance of pore formation on the plasma membrane.
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http://dx.doi.org/10.1242/jcs.204206DOI Listing
October 2017

The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells.

Biol Cell 2017 Nov 6;109(11):375-380. Epub 2017 Sep 6.

Heidelberg University Biochemistry Center, Heidelberg, Germany.

Fibroblast Growth Factor 2 (FGF2) is a potent cell survival factor involved in tumour-induced angiogenesis. FGF2 is secreted from cells through an unconventional secretory mechanism based upon direct translocation across the plasma membrane. The molecular mechanism underlying this process depends on a surprisingly small set of trans-acting factors that are physically associated with the plasma membrane. FGF2 membrane translocation is mediated by the ability of FGF2 to oligomerise and to insert into the plasma membrane in a PI(4,5)P -dependent manner. Membrane-inserted FGF2 oligomers are dynamic translocation intermediates that are disassembled at the extracellular leaflet mediated by membrane proximal heparan sulphate proteoglycans. This process results in the exposure of FGF2 on cell surfaces as part of its unconventional mechanism of secretion. Although the trans-acting factors and cis-elements in FGF2 required for unconventional secretion have been known for a while, the core mechanism of this mysterious process has now been reconstituted with purified components establishing the molecular basis of FGF2 secretion from tumour cells.
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http://dx.doi.org/10.1111/boc.201700036DOI Listing
November 2017

Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components.

Elife 2017 07 19;6. Epub 2017 Jul 19.

Heidelberg University Biochemistry Center, Heidelberg, Germany.

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.
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http://dx.doi.org/10.7554/eLife.28985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601999PMC
July 2017

Tyrosine Kinase Expressed in Hepatocellular Carcinoma, TEC, Controls Pluripotency and Early Cell Fate Decisions of Human Pluripotent Stem Cells via Regulation of Fibroblast Growth Factor-2 Secretion.

Stem Cells 2017 09 6;35(9):2050-2059. Epub 2017 Jul 6.

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.
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http://dx.doi.org/10.1002/stem.2660DOI Listing
September 2017

Bioinspired carbide-derived carbons with hierarchical pore structure for the adsorptive removal of mercury from aqueous solution.

Chem Commun (Camb) 2017 Apr;53(35):4845-4848

Department of Bioanalytic Chemistry, Dresden University of Technology, Bergstraße 66, D-01062, Dresden, Germany.

Biosilica of the diatom species Thalassiosira pseudonana is used as hard template for the synthesis of silicon carbide-derived carbons. The typical species-specific macroporous structure is retained during the nanocasting-chlorine treatment process and the resulting materials exhibit very high specific surface areas up to 2300 m g. Bioinspired carbons show very high capacities in mercury adsorption from aqueous solution compared to reference materials.
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http://dx.doi.org/10.1039/c6cc08041cDOI Listing
April 2017

Illuminating solid gas storage in confined spaces - methane hydrate formation in porous model carbons.

Phys Chem Chem Phys 2016 Jul;18(30):20607-14

Laboratorio de Materiales Avanzados, IUMA, Universidad de Alicante, Ctra. San Vicente del Raspeig-Alicante s/n, E-03690 San Vicente del Raspeig, Spain.

Methane hydrate nucleation and growth in porous model carbon materials illuminates the way towards the design of an optimized solid-based methane storage technology. High-pressure methane adsorption studies on pre-humidified carbons with well-defined and uniform porosity show that methane hydrate formation in confined nanospace can take place at relatively low pressures, even below 3 MPa CH4, depending on the pore size and the adsorption temperature. The methane hydrate nucleation and growth is highly promoted at temperatures below the water freezing point, due to the lower activation energy in ice vs. liquid water. The methane storage capacity via hydrate formation increases with an increase in the pore size up to an optimum value for the 25 nm pore size model-carbon, with a 173% improvement in the adsorption capacity as compared to the dry sample. Synchrotron X-ray powder diffraction measurements (SXRPD) confirm the formation of methane hydrates with a sI structure, in close agreement with natural hydrates. Furthermore, SXRPD data anticipate a certain contraction of the unit cell parameter for methane hydrates grown in small pores.
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http://dx.doi.org/10.1039/c6cp03993fDOI Listing
July 2016

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2.

J Biol Chem 2016 08 5;291(34):17787-803. Epub 2016 Jul 5.

From the Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany,

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.
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http://dx.doi.org/10.1074/jbc.M116.729384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016171PMC
August 2016

Promoting Patient and Family Partnerships in Ambulatory Care Improvement: A Narrative Review and Focus Group Findings.

Adv Ther 2016 08 28;33(8):1417-39. Epub 2016 Jun 28.

Group Health Research Institute, Seattle, WA, USA.

Introduction: Ambulatory practices that actively partner with patients and families in quality improvement (QI) report benefits such as better patient/family interactions with physicians and staff, and patient empowerment. However, creating effective patient/family partnerships for ambulatory care improvement is not yet routine. The objective of this paper is to provide practices with concrete evidence about meaningfully involving patients and families in QI activities.

Methods: Review of literature published from 2000-2015 and a focus group conducted in 2014 with practice advisors.

Results: Thirty articles discussed 26 studies or examples of patient/family partnerships in ambulatory care QI. Patient and family partnership mechanisms included QI committees and advisory councils. Facilitators included process transparency, mechanisms for acting on patient/family input, and compensation. Challenges for practices included uncertainty about how best to involve patients and families in QI. Several studies found that patient/family partnership was a catalyst for improvement and reported that partnerships resulted in process improvements. Focus group results were concordant.

Conclusion: This paper describes emergent mechanisms and processes that ambulatory care practices use to partner with patients and families in QI including outcomes, facilitators, and challenges.

Funding: Gordon and Betty Moore Foundation.
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http://dx.doi.org/10.1007/s12325-016-0364-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969329PMC
August 2016

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

PLoS One 2016 21;11(4):e0153009. Epub 2016 Apr 21.

Heidelberg University Biochemistry Center, Heidelberg, Germany.

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153009PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839656PMC
February 2017

Inflammasome-dependent IL-1β release depends upon membrane permeabilisation.

Cell Death Differ 2016 07 12;23(7):1219-31. Epub 2016 Feb 12.

Grupo de Inflamación Molecular, Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas, Hospital Clínico Universitario Virgen de la Arrixaca, Instituto Murciano de Investigación Biosanitaria (IMIB-Arrixaca), Murcia, Spain.

Interleukin-1β (IL-1β) is a critical regulator of the inflammatory response. IL-1β is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1β release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1β, we have discovered that the secretion of IL-1β after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1β in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1β release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.
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http://dx.doi.org/10.1038/cdd.2015.176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946890PMC
July 2016

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand.

J Biol Chem 2015 Nov 28;290(45):27015-27020. Epub 2015 Sep 28.

Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany. Electronic address:

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P2-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.
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http://dx.doi.org/10.1074/jbc.R115.689257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646381PMC
November 2015

Synthesis of Ordered Mesoporous Carbon Materials by Dry Etching.

Chemistry 2015 Oct 26;21(42):14753-7. Epub 2015 Aug 26.

Department of Inorganic Chemistry, TU Dresden, Bergstrasse 66, 01069 Dresden (Germany), Fax: (+49) 351-463-37287.

A novel synthesis method for ordered mesoporous carbons is presented. The inverse replication of a silica template was achieved using the carbonization of sucrose within mesoporous KIT-6. Instead of liquid acid etching, as in classical nanocasting, a novel dry chlorine etching procedure for template removal is presented for the first time. The resultant ordered mesostructured carbon material outperforms carbons obtained by conventional hard templating with respect to high specific micro- and mesopore volumes (0.6 and 1.6 cm(3) g(-1) , respectively), due to the presence of a hierarchical pore system. A high specific surface area of 1671 m(2) g(-1) was achieved, rendering this synthesis route a highly convenient method to produce ordered mesoporous carbons.
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http://dx.doi.org/10.1002/chem.201502038DOI Listing
October 2015

A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

J Proteome Res 2015 Aug 20;14(8):3229-38. Epub 2015 Jul 20.

#Department of Functional Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.

Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.
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http://dx.doi.org/10.1021/acs.jproteome.5b00199DOI Listing
August 2015

HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion.

J Biol Chem 2015 Sep 16;290(36):21976-84. Epub 2015 Jul 16.

From the Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany and

HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel β sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.
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http://dx.doi.org/10.1074/jbc.M115.667097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571951PMC
September 2015

Advanced structural analysis of nanoporous materials by thermal response measurements.

Langmuir 2015 Apr 25;31(13):4040-7. Epub 2015 Mar 25.

†Department of Inorganic Chemistry, Dresden University of Technology, Bergstrasse 66, D-01062 Dresden, Germany.

Thermal response measurements based on optical adsorption calorimetry are presented as a versatile tool for the time-saving and profound characterization of the pore structure of porous carbon-based materials. This technique measures the time-resolved temperature change of an adsorbent during adsorption of a test gas. Six carbide and carbon materials with well-defined nanopore architecture including micro- and/or mesopores are characterized by thermal response measurements based on n-butane and carbon dioxide as the test gases. With this tool, the pore systems of the model materials can be clearly distinguished and accurately analyzed. The obtained calorimetric data are correlated with the adsorption/desorption isotherms of the materials. The pore structures can be estimated from a single experiment due to different adsorption enthalpies/temperature increases in micro- and mesopores. Adsorption/desorption cycling of n-butane at 298 K/1 bar with increasing desorption time allows to determine the pore structure of the materials in more detail due to different equilibration times. Adsorption of the organic test gas at selected relative pressures reveals specific contributions of particular pore systems to the increase of the temperature of the samples and different adsorption mechanisms. The use of carbon dioxide as the test gas at 298 K/1 bar provides detailed insights into the ultramicropore structure of the materials because under these conditions the adsorption of this test gas is very sensitive to the presence of pores smaller than 0.7 nm.
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http://dx.doi.org/10.1021/acs.langmuir.5b00490DOI Listing
April 2015