On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release.

The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin. This complex is disassembled by N-ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18-1 and Munc13s organize SNARE complex assembly in an NSF-SNAP-resistant manner. Synaptotagmin-1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane-binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re-interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses.

Dynamics of Dystrophin's Actin-Binding Domain.

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

Structural Impact of Phosphorylation and Dielectric Constant Variation on Synaptotagmin's IDR.

We used time-resolved Förster resonance energy transfer, circular dichroism, and molecular dynamics simulation to investigate the structural dependence of synaptotagmin 1's intrinsically disordered region (IDR) on phosphorylation and dielectric constant. We found that a peptide corresponding to the full-length IDR sequence, a ∼60-residue strong polyampholyte, can sample structurally collapsed states in aqueous solution, consistent with its κ-predicted behavior, where κ is a sequence-dependent parameter that is used to predict IDR compaction. In implicit solvent simulations of this same sequence, lowering the dielectric constant to more closely mimic the environment near a lipid bilayer surface promoted further sampling of collapsed structures. We then examined the structural tendencies of central region residues of the IDR in isolation. We found that the exocytosis-modulating phosphorylation of Thr112 disrupts a local disorder-to-order transition induced by trifluoroethanol/water mixtures that decrease the solution dielectric constant and stabilize helical structure. Implicit solvent simulations on these same central region residues testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric. In these helical conformers, lysine-aspartic acid salt bridges contribute to stabilization of transient secondary structure. In contrast, phosphorylation results in formation of salt bridges unsuitable for helix formation. Collectively, these results suggest a model in which phosphorylation and compaction of the IDR sequence regulate structural transitions that in turn modulate neuronal exocytosis.

Structural basis for high-affinity actin binding revealed by a ß-III-spectrin SCA5 missense mutation.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein ß-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which ß-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.

Synaptotagmin I's Intrinsically Disordered Region Interacts with Synaptic Vesicle Lipids and Exerts Allosteric Control over C2A.

Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein that senses the calcium ion (Ca(2+)) influx to trigger fast synchronous release of neurotransmitter. How the cytosolic domains of Syt I allosterically communicate to propagate the Ca(2+) binding signal throughout the protein is not well understood. In particular, it is unclear whether the intrinsically disordered region (IDR) between Syt I's transmembrane helix and first C2 domain (C2A) plays an important role in allosteric modulation of Ca(2+) binding. Moreover, the structural propensity of this IDR with respect to membrane lipid composition is unknown. Using differential scanning and isothermal titration calorimetry, we found that inclusion of the IDR does indeed allosterically modulate Ca(2+) binding within the first C2 domain. Additionally through application of nuclear magnetic resonance, we found that Syt I's IDR interacts with membranes whose lipid composition mimics that of a synaptic vesicle. These findings not only indicate that Syt I's IDR plays a role in regulating Syt I's Ca(2+) sensing but also indicate the IDR is exquisitely sensitive to the underlying membrane lipids. The latter observation suggests the IDR is a key route for communication of lipid organization to the adjacent C2 domains.

Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Allostery and instability in the functional plasticity of synaptotagmin I.

Synaptotagmin I (Syt I) is the calcium ion sensor for regulated release of neurotransmitter. How Syt I mediates this cellular event has been a question of extensive study for decades and yet, a clear understanding of the protein's diverse functionality has remained elusive. Using tools of thermodynamics, we have identified two intrinsic properties that may account for Syt I's functional plasticity: marginal stability and negative coupling. These two intrinsic properties have the potential to provide great conformational flexibility and suggest that Syt I's functional plasticity stems in part from subtle rearrangements in the protein's conformational ensemble. This model for Syt I function is discussed within the context of the nervous system's overall plasticity.

Negative coupling as a mechanism for signal propagation between C2 domains of synaptotagmin I.

Synaptotagmin I (Syt I) is a vesicle-localized protein implicated in sensing the calcium influx that triggers fast synchronous release of neurotransmitter. How Syt I utilizes its two C2 domains to integrate signals and mediate neurotransmission has continued to be a controversial area of research, though prevalent hypotheses favor independent function. Using differential scanning calorimetry and fluorescence lifetime spectroscopy in a thermodynamic denaturation approach, we tested an alternative hypothesis in which both domains interact to cooperatively disseminate binding information. The free energy of stability was determined for C2A, C2B, and C2AB constructs by globally fitting both methods to a two-state model of unfolding. By comparing the additive free energies of C2A and C2B with C2AB, we identified a negative coupling interaction between the C2 domains of Syt I. This interaction not only provides a mechanistic means for propagating signals, but also a possible means for coordinating the molecular events of neurotransmission.

Unicommissural aortic valves: gross, histological, and immunohistochemical analysis of 52 cases (1978-2008).

BACKGROUND: Unicommissural aortic valves (UAVs) are rare anomalies in which adjacent cusps of two commissures are congenitally fused. Currently, features of UAVs are poorly characterized. METHODS: Fifty-two surgical and autopsy cases of UAV at Mayo Clinic were evaluated for various clinicopathologic features. Histology and immunohistochemistry (IHC) were used in 30 UAVs to identify biomarkers important to developing aortic stenosis. RESULTS: In 52 UAV patients (58% were male, 42% were female), their ages ranged from 18 weeks' gestation to 80 years (mean, 28 years). Functional status was pure stenosis in 20, pure regurgitation in 9, combined in 22, and normal in 1. Common additional cardiovascular disorders included left ventricular hypertrophy (56%) and ascending aortopathies (42%). The position of the true commissure was determined in 30 UAVs and was left posterior in 73%. Gross calcification increased exponentially with age, starting as early as 16 years. Microscopically, the values of the 3 youngest patients showed dysplasia. Other UAVs exhibited fibrous thickening (93%), ventricular pads (89%), aortic pads (81%), and thickened spongy layer (74%). Macrophages were the most common leukocyte by IHC. Bone morphogenetic protein-2 was positive in 27 IHC cases; osteopontin was positive in 15, and matrix metalloproteinase (MMP) 2, MMP-9, and MMP-14 were positive in 1, 6, and 4 cases, respectively. CONCLUSION: The functional status of UAVs typically involves stenosis but can vary in type and degree or rarely be normal. In early stenosis (<16 years), the pathology is primarily fibrosis with minimal calcification. UAVs show more calcification compared with age-matched bicuspid or tricuspid aortic valves. The molecular mechanisms of calcification and fibrosis in UAVs remain incompletely understood.

Prosthetic valve endocarditis: clinicopathological correlates in 122 surgical specimens from 116 patients (1985-2004).

BACKGROUND: Few studies have documented the clinicopathological features of prosthetic valve endocarditis independently of native valve endocarditis. STUDY DESIGN: Retrospective analysis of patients undergoing cardiac surgery for prosthetic valve endocarditis at our institution (1985-2004). METHODS: Medical records and microscopic slides were reviewed from 116 patients for demographics, infecting organisms, comorbidities, and pathologic features. RESULTS: Patients were 12-86 years old (mean, 59 years). Among 122 valves, 64% were from men and 67% were purely regurgitant. Aortic prosthetic valve endocarditis frequently affected men (76%); mitral prosthetic valve endocarditis often affected women (62%). Embolization occurred in 35% and heart failure in 32%. Prevalent predisposing conditions were the prosthetic valve alone (43%) and diabetes mellitus (20%). Prosthetic valve endocarditis was aortic or mitral in 98% and was active in 70%. Annular abscess or paravalvular leak affected mechanical valves more frequently than bioprosthetic (89% vs. 65%; P=.001). Causative organisms (n=116) included Staphylococcus aureus (30%), coagulase-negative staphylococcus (22%), viridans streptococci (18%), enterococci (10%), other streptococci (8%), and other organisms (12%). S. aureus was the most prevalent cause of early-onset (38%) and late-onset (30%) prosthetic valve endocarditis. Coagulase-negative staphylococcus caused early-onset (31%) and most intermediate-onset (40%) disease and had a shorter median implantation-to-infection time than other organisms (6.5 vs. 61.3 months; P<.001). Viridans streptococci and enterococci primarily caused late-onset endocarditis. For active infections by cocci, most cases exhibited strong Gram staining, but four showed only strong Grocott methenamine silver staining. CONCLUSIONS: Cocci accounted for 83% of infections. Early-onset prosthetic valve endocarditis was primarily staphylococcal, and late-onset prosthetic valve endocarditis resembled native valve endocarditis. Both Gram and Grocott methenamine silver stains were necessary to reliably identify organisms microscopically.

MGMT immunoexpression in silent subtype 3 pituitary adenomas: possible therapeutic implications.

The objective of the study was to assess O(6)-methylguanine-DNA methyltransferase (MGMT) immunoreactivity in pituitary adenomas of silent subtype 3 as a potential indicator of temozolomide susceptibility. The Mayo Clinic Anatomic Pathology Database was searched for all cases of silent subtype 3 pituitary adenoma. Each of the 23 cases identified had been confirmed on the basis of histology, immunohistochemical staining for pituitary hormones, as well as on diagnostic ultrastructural criteria. Unstained microscopic sections were immunostained for MGMT and were semiquantitatively assessed. Of the 23 tumors examined, 18 (78%) showed no MGMT immunoreactivity. The remaining five (22%) showed immunoreactivity in < or =50% of tumor cells. Among eight of the most clinically aggressive tumors in this study, six (75%) lacked MGMT immunoreactivity. The observed lack of or low-level expression of MGMT by this distinctive, clinically aggressive form of pituitary adenoma suggests potential efficacy of treatment with the alkylating agent temozolomide.

Echocardiographic features of cardiac amyloidosis presenting as endomyocardial disease in a 54-year-old male.

Although primary cardiac amyloidosis is a rare affliction of the heart, its clinical and pathology features have been well described. Patients generally present with exertional dyspnea, chest discomfort, or congestive heart failure. Cardiac imaging typically reveals ventricular wall thickening, systolic and diastolic dysfunction, valvular thickening, and pericardial effusion. In the case reported herein, a patient with suspected cardiac amyloid demonstrated electrocardiographic and echocardiographic findings that were not only inconsistent with typical depositional patterns, but also potentially suggestive of endomyocardial disease.

Congenital aortic sinus aneurysm causing sudden unexpected death in a 56-year-old woman.

Sudden unexpected death due to hemopericardium generally has 2 main causes, rupture of the ascending aorta or rupture of a transmural myocardial infarct. We report an unusual cause of fatal hemopericardium in a 56-year-old woman that occurred in the setting of a motor vehicle mishap.At autopsy, 300 mL of blood were present in the pericardial sac, due to rupture of a previously undetected congenital aortic sinus aneurysm. The low velocity of the automobile at the time of impact with a fence, and the absence of vehicle-related trauma, led to the conclusion that rupture of the aneurysm caused the motor vehicle mishap, rather than vice versa. Because of the typically silent nature of such aneurysms, it is likely that their rupture will occur out-of-hospital and require a forensic investigation.

Neuronal ceroid lipofuscinosis in a 31-year-old woman presenting as biventricular heart failure with restrictive features.

A 31-year-old woman presented with dyspnea and left-sided chest discomfort and was found to have biventricular heart failure with impaired ventricular filling. Clinically, she was thought to have restrictive cardiomyopathy or constrictive pericarditis. Transmission electron microscopy of myocardial tissue unexpectedly revealed crosshatched, curvilinear, and fingerprint depositions, which were characteristic for neuronal ceroid lipofuscinosis. Cardiac involvement by this inherited disorder is discussed in light of the findings in this patient and in 15 other reported cases.

Complications of endovascular polymers associated with vascular introducer sheaths and metallic coils in 3 patients, with literature review.

BACKGROUND: Hydrophilic coatings on arterial introducer sheaths reduce the frequency of spasm during cardiac catheterization. Moreover, during treatment of cerebral artery aneurysms, hydrophilic coatings on microcatheters ease their maneuverability, and polymers within embolization coils enhance neointimal fibrosis at the aneurysm neck. Foreign-body giant cell reactions can result when a polymer is stripped from its source. CASES 1 AND 2: A 51-year-old man and a 66-year-old woman underwent coronary angiography via the radial artery, using hydrophilic-coated sheaths. They both developed tender lesions at the access site 2 weeks later. Microscopy of resected lesions showed pieces of hydrophilic coating and granulomatous inflammation, as has been reported by others. CASE 3: A 58-year-old woman underwent embolization of a ruptured distal right internal carotid artery aneurysm using polymer-containing coils. Nine months later, she began developing multiple right-sided cerebral ring-enhancing lesions. Biopsy revealed granulomas and microabscesses, in which polymer filaments were later identified. To our knowledge, this complication has not been described previously. CONCLUSIONS: Hydrophilic coating may dislodge and induce a prominent foreign-body granulomatous response or microabscesses. Although the culprit radial artery sheath is now rarely used, embolization coils containing polymers are commonly deployed in clinical practice and may be a source of recurrent inflammatory lesions.

Hamartomas of mature cardiac myocytes: report of 7 new cases and review of literature.

Only 8 cases of hamartomas of mature cardiac myocytes have been reported. The aim of the study was to describe 7 new cases and provide clinicopathologic correlation. Our anatomical pathology database was searched for all cases of cardiac hamartoma, of which 7 represented mature myocyte type. Medical records were reviewed for clinical information, and microscopic slides were evaluated for extent of characteristics. Five males and 2 females ranged in age from 6 months to 74 years (mean, 23 years). There were 11 ventricular hamartomas (8 left free wall, 2 right free wall, 1 septum). Death in 3 infants was unrelated to incidental hamartomas discovered at autopsy. A 10- and 16-year-old were asymptomatic but had abnormal electrocardiogram (ECG) results, which led to detection of cardiac masses by imaging studies. Two adult males had only mild coronary disease angiographically. The 57-year-old, who died suddenly, had a 7-year history of abnormal ECG results. The 74-year-old, who died after aortic surgery, had a 3-year history of chest discomfort. Their hamartomas were identified at autopsy and contributed to sudden death in 1. Microscopically, all hamartomas were involved by myocyte hypertrophy and disarray, without inflammation or calcification. Myocyte vacuolization and venular dilatation occurred only in the pediatric cases, and interstitial adipose tissue only affected 1 adult. In conclusion, hamartomas of mature cardiac myocytes may be detected at any age. They primarily affect males, arise predominantly in the left ventricle, are asymptomatic, may have nonspecific ECG findings, and rarely may be associated with sudden death. Microscopic findings in infants differ from older patients.

Beta-catenin mutations do not contribute to cardiac fibroma pathogenesis.

Cardiac fibromas are the 2nd most common benign cardiac tumor occurring in children and bear a striking morphologic resemblance to soft tissue or desmoid fibromatosis. Since activating mutations in beta-catenin are common in desmoid fibromatosis as well as other spindle cell proliferations, the aim of our study was to determine if such mutations could be identified in cardiac fibroma. Nine cardiac fibromas from patients with surgical resection were examined for beta-catenin mutations by immunoperoxidase staining for beta-catenin protein and DNA sequencing of a region in exon 3 of the beta-catenin gene, where relatively conserved mutations have been described in desmoid fibromatosis. The mean age of the patients was 7.6 years (range: 10 weeks to 27 years), and 6 of the patients were male. No nuclear staining for beta-catenin was seen in the fibroma cells by immunoperoxidase methods. The beta-catenin exon 3 sequence data showed no mutations in any of the 9 tumors. We conclude that despite their morphologic similarity, cardiac fibroma and desmoid fibromatosis do not share this common molecular pathway of neoplastic growth.

Recurrent cardiac calcific amorphous tumor: the CAT had a kitten.

BACKGROUND: Cardiac calcified amorphous tumors (CATs) represent degenerating intracavitary mural thrombi that can mimic neoplasms and cause symptoms due to embolization or obstruction. Surgical excision is generally curative. Postoperative recurrences have not been previously reported. METHODS: Medical, surgical, and pathological records were reviewed in a patient who had undergone removal of a recurrent cardiac CAT. RESULTS: The patient, now a 23-year-old woman, had undergone excision of the initial right ventricular mass on November 4, 2003, because of recent pulmonary embolization. Extensive clinical evaluation showed no coagulation abnormality. Follow-up postoperative echocardiograms showed incomplete excision and subsequent enlargement. As a result, the recurrent mass was excised on March 14, 2006. Microscopic evaluation showed degenerating and focally calcifying thrombus, without neoplastic features. CONCLUSION: Cardiac CAT may recur and enlarge following surgical excision. Periodic postoperative follow-up with cardiac imaging studies may be indicated, particularly if there is evidence of incomplete excision.

Primary cardiac amyloidosis with 20-year survival.

BACKGROUND: The natural history of primary amyloidosis is poor, and for patients with symptomatic cardiac involvement, survival is generally less than 6 months. Even among treated patients with amyloid heart disease, survival beyond 5 years is rare. CASE REPORT: We report a patient with primary cardiac amyloidosis who is currently alive 20 years after his initial diagnosis. The extent and subtype of amyloid were documented by endomyocardial biopsy both at the time of initial diagnosis and 20 years later. To our knowledge, this is the longest survival ever reported for a patient with cardiac involvement by primary amyloidosis. CONCLUSION: The remarkably long stabilization of amyloid deposition in this patient may be attributed to early diagnosis, early institution of therapy, and, possibly, favorable genetic factors.