Ratpost: a searchable database of protein-inactivating sequence variations in 40 sequenced rat-inbred strains

Mammalian Genome - Rat-inbred strains are essential as scientific tools. We have analyzed the publicly available genome sequences of 40 rat-inbred strains and provide an overview of sequence...     

Diffusion of kinesin motors on cargo can enhance binding and run lengths during intracellular transport

Cellular cargoes, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargoes with rigidly attached motors, in contrast to many biological cargoes that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical three-dimensional computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargoes with clustered motors are transported efficiently but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly but are transported inefficiently. Finally, cargoes with freely diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.     

Age-dependency,climate, and environmental controls of recent tree growth trends at subarctic and alpine treelines

Spatial and temporal variability in growth and climate response of trees at and near treeline was investigated in the western Mackenzie Mountains, Northwest Territories, and the Hudson Bay Lowlands of northern Manitoba. Residual ring width chronologies were constructed using cores extracted from 108 trees in the mountains and 170 from the lowlands, and compared to historical climate data. Growth of most trees exhibited significant correlations with summer and autumn temperatures, and the growth–climate relationship did not differ noticeably between trees at and distal to treeline. Most mountain trees had significant positive growth trends from 1851 to 2006 that corresponded with warming over the same period, while growth trends varied among sites and species in the lowlands. Regionally, growth of all species responded positively to warming during the 20th century with the exception of lowland Picea mariana, which exhibited little response. Growth response for most trees was age-dependent, with trees established after 1920 demonstrating improved growth and sensitivity to temperature than older individuals, and growth of most species since the 1990s was greater than any time during the last 250 years, particularly for lowland Larix laricina. This study suggests that site factors and tree age can be more important drivers of local-scale growth trends than regional climate at arctic treelines where temperature is often assumed to be the main constraint on tree growth.     

Association of CAT polymorphisms with catalase activity and exposure to environmental oxidative stimuli

We tested the hypotheses that catalase activity is modified by CAT single nucleotide polymorphisms (SNPs) (-262;-844), and by their interactions with oxidant exposures (coal dusts, smoking), lymphotoxin alpha (LTA, NcoI) and tumor necrosis factor (TNF, -308) in 196 miners. Erythrocyte catalase, superoxide dismutase, and glutathione peroxidase activities were measured. The CAT -262 SNP was related to lower catalase activity (104, 87 and 72 k/g hemoglobin for CC, CT and TT, respectively, p < 0.0001). Regardless of CAT SNPs, the LTA NcoI but not the TNF-308 SNP was associated with catalase activity (p = 0.04 and p = 0.8). CAT -262 T carriers were less frequent in highly exposed miners (OR = 0.39 [0.20–0.78], p = 0.007). In CAT -262 T carriers only, catalase activity decreased with high dust exposure (p = 0.01). Haplotype analyses (combined CAT SNPs) confirm these results. Results show that CAT -262 and LTA NcoI SNPs, and interaction with coal dust exposure, influenced catalase activity.     

Conservative Treatment for Postprostatectomy Incontinence

Postprostatectomy incontinence (PPI) is a bothersome complication of radical prostatectomy. Although most men recover from PPI, some men continue to have persistent urinary incontinence. The initial management of persistent PPI usually consists of conservative measures such as pelvic floor muscle exercises. Surgical treatments are usually not entertained for men with urinary incontinence until conservative treatments have failed. This article discusses risk factors for PPI and various options for its treatment, including biofeedback and pharmacotherapy.Key words: Postprostatectomy incontinence, Radical prostatectomy, Pelvic floor muscle training, Pelvic floor stimulationPostprostatectomy incontinence (PPI) is a bothersome complication of radical prostatectomy (RP). Although most men recover from PPI, some men continue to have persistent urinary incontinence and roughly half of these men seek treatment.¹ It is important to understand the natural history of postprostatectomy urinary dysfunction prior to initiating treatment. Generally, the initial management of persistent PPI consists of conservative measures such as pelvic floor muscle exercises.     

A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion.The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics. An effective method for the detection of posttranslational modifications of proteins is the metabolic incorporation of modified biomolecules such as tagged carbohydrates or lipids (1). Reversible interactions of enzyme inhibitors, natural products, or drugs can be detected when one appends photocrosslinking agents, thereby facilitating target discovery (2, 3). A particularly interesting example of protein labeling is activity-based protein profiling (ABPP)¹ (4, 5), which utilizes the intrinsic catalytic activity of a target enzyme for the covalent attachment of an affinity or visualization tag. ABPP makes use of small molecules (activity-based probes (ABPs)) that react with the active form of a specific enzyme or enzyme class by means of a “warhead,” which is often derived from a mechanism-based enzyme inhibitor (Fig. 1A). DCG-04, for example, is based on the naturally occurring inhibitor E-64 and targets the papain family of cysteine proteases via covalent attachment of the epoxysuccinate group to the active site cysteine (Fig. 1B) (6).Open in a separate windowFig. 1.The cleavable linker strategy in ABPP. A, the elements of an ABP. B, the example ABP DCG-04, an epoxysuccinate-containing probe for clan CA cysteine proteases. DCG-04 is based on the naturally occurring protease inhibitor E-64. C, schematic strategy of cleavable linker-mediated target identification. D, the cleavage mechanism of a vicinal diol.Bulky fluorophore or biotin tags on chemical probes might interfere with efficient protein binding. Moreover, they can negatively influence the cell permeability of probes, which therefore limits their applicability in in vitro experiments. Bioorthogonal chemistries, such as the Bertozzi-Staudinger ligation (7) and the 1,3-bipolar cycloaddition of an azide and an alkyne (click chemistry) (8), allow tandem labeling strategies in which a biotin or a fluorophore is attached to an enzyme probe complex in a separate step. Consequently, the probes themselves only carry azide or alkyne groups as “mini-tags.” Tandem labeling using bioorthogonal chemistry has now become a widely used strategy to label biomolecules in lysates and in live cells (9–11).An essential step in ABPP, as well as in other chemical proteomics approaches, is the elucidation of the tagged proteins. This usually involves a biotin-mediated enrichment step followed by mass-spectrometry-based identification. Although the streptavidin-biotin interaction allows efficient enrichment as a result of the strong binding affinity (K_d ∼ 10⁻¹⁵ m), it also has limitations. The quantitative elution of biotinylated proteins requires harsh conditions (12), which lead to contamination of the sample by endogenous biotinylated and non-specifically bound proteins. These other proteins will be identified together with the real protein targets. Given that subsequent target validation with secondary assays can be a costly and time-consuming process, a reduction in false positive identifications is highly desirable. For cleaner protein identification, cleavable linker strategies (13) that allow the selective release of target proteins have been developed (Fig. 1C). The commercially available disulfide linker can be cleaved under mild conditions, but it suffers from premature cleavage in reducing media such as the intracellular environment and reducing buffers used for click chemistry and in vitro reactions of cysteine proteases. Therefore, a variety of alternative linkers for proteomics applications have been reported, including a sterically hindered disulfide (14), diazobenzenes (15–19), hydrazones (20, 21), silanes (22), light sensitive linkers (23–25), tobacco etch virus protease sensitive linkers (26, 27), and a levulinoyl-based linker (28). The synthesis of some of these linkers is lengthy or difficult to scale up, which limits their general application in chemical proteomics.Ideally, a cleavable linker is stable under a wide variety of conditions, is efficiently and selectively cleaved, and can be synthesized in a low number of easy chemical transformations. We aimed to meet these requirements by using a vicinal diol as a cleavable linker system. When vicinal diols are treated with sodium periodate (NaIO₄), the carbon–carbon bond is cleaved (Fig. 1D). Periodate treatment of proteins can result in side-reactions, such as the cleavage of linked carbohydrates or the oxidation of N-terminal serine and threonine residues. However, these N-termini rarely occur in proteins and are therefore of minor concern. In general, the mild, neutral conditions of periodate cleavage are compatible with proteins. This has been illustrated in the past, for example, by its application in the detection of protein–protein interactions (29) and the creation of unliganded MHC class I molecules (30). In this article, we report the chemical proteomics application of diol cleavable linker probes. We show that the synthesis of the linker and its probe derivatives is straightforward, that the linker is compatible with tandem click labeling, that enrichment and release of probe targets is efficient, and that the identification of targets takes place with significantly lower background than in on-bead digestion protocols.     

Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ∼40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.Smith-Lemli-Opitz syndrome (SLOS)¹ is an autosomal recessive disorder associated with subnormal growth and failure to thrive, mental retardation and neurosensory deficits, and multiple congenital anomalies, including dysmorphologies (1, 2). Early epidemiological studies estimated the incidence of SLOS as 1 in 20,000 to 1 in 60,000 live births, primarily among Caucasians (1, 2). However, more recent studies suggest that the SLOS carrier frequency is ∼1 in 30 to 1 in 50; this predicts a much higher actual disease frequency, ranging from 1 in 1,590 to 1 in 17,000 (3, 4), making SLOS the fourth most common autosomal recessive human disease (after cystic fibrosis, phenylketonuria, and hemochromatosis). Mutation of the DHCR7 gene is the intrinsic cause of SLOS; this gene encodes the enzyme DHCR7 (3β-hydroxysterol-Δ7-reductase, a.k.a. 7-dehydrocholesterol reductase; EC1.3.1.21), which catalyzes the final step in the cholesterol biosynthetic pathway, reducing the Δ7 double bond and thus converting 7-dehydrocholesterol (7DHC) to cholesterol (4, 5). As a consequence, markedly reduced levels of cholesterol and aberrantly elevated levels of the cholesterol precursor 7DHC (and its epimer, 8DHC) are observed in the majority of affected SLOS patients (6, 7). Therefore, the clinical suspicion of SLOS is confirmed by elevated 7DHC in plasma or tissues, typically demonstrated via chromatographic methods (e.g. HPLC or GC/MS) (8, 9).Visual capacity may become compromised in SLOS patients because of a variety of congenital or postnatal pathologies, such as cataracts, aniridia, corneal endothelium defects, sclerocornea, electrophysiological defects in the retina, optic nerve abnormalities, or other ophthalmologic problems (10, 11). We currently lack full knowledge of the exact pathobiological mechanism underlying SLOS, but additional insights may be afforded by studies employing a rodent model of the disease in which rats are treated with AY9944 (trans-1,4-bis[2-chlorobenzylaminomethyl] cyclohexane dihydrochloride), a relatively selective inhibitor of DHCR7 (12–14). We previously described progressive retinal degeneration in this rat model of SLOS, which is characterized by the shortening of retinal rod outer segments, pyknosis and thinning of the outer nuclear layer (ONL) of the retina (which contains the photoreceptor nuclei), and accumulation of membranous/lipid inclusions in the retinal pigment epithelium (RPE) (12, 13). Reduced rod outer segment membrane fluidity, primarily caused by a dramatic (30 to 40 mol%) decline in docosahexaenoic acid (22:6, n3) levels relative to age-matched controls, also was observed in the SLOS rat model by three postnatal months (15, 16). Retinal function and sterol steady-state in the same rat model of SLOS can be partially rescued using a high-cholesterol diet (2% by weight), although histological degeneration of the retina still occurs (17). However, the molecular mechanisms that underlie the observed electrophysiological defects in the retina, the accumulation of membranous/lipid inclusions in the RPE, the shortening of retinal rod outer segments, and the initiation of ONL pyknosis in the SLOS rat model remain poorly understood. Therefore, a comprehensive profiling of the retinal proteomes of AY9944-treated versus age-matched untreated control rats may contribute to further understanding of the underlying mechanisms responsible for the retinopathy associated with the SLOS model and, by extension, the human disease.Nevertheless, extensive and reliable expression profiling of the retinal proteome remains a prominent challenge, owing to the need to quantify data from multiple animals and a high percentage of integral membrane and membrane-associated proteins (18, 19). Label-free approaches can compare multiple replicates (20–22) with quantitative accuracy comparable to that attained with stable isotope-labeling methods (23–25). However, in order to achieve reliable relative quantification, highly quantitative and reproducible sample preparation and LC/MS analysis are required for relatively large-scale sample cohorts.In the present study, we performed a reproducible, well-controlled, ion-current-based comparative proteomic analysis of the retinas from AY9944-treated versus age/sex-matched control rats (n = 5 animals per group). A high-concentration detergent mixture was used for the efficient extraction of proteins from retinas, and samples then underwent a reproducible precipitation/on-pellet-digestion procedure and long-column, 7-h nano-LC-MS analysis. These approaches ensured extensive comparative analysis of retina samples with 10 animals. The preparative and analytical procedures were carefully optimized and controlled to ensure optimal reproducibility. Two label-free approaches, the ion-current-based method and a spectral counting method, were compared in parallel. The altered proteins were subjected to functional annotation, and selected groups of proteins of interest were further validated by means of Western blot and correlative immunohistochemical analysis.