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Jennifer L Martin

Publications and source records attributed to Jennifer L Martin.

At least 37 records · Page 2Linked to original sources
PubMed2005

Structural, mutagenic, and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase.

The X-ray structure of human phenylethanolamine N-methyltransferase (hPNMT) complexed with its product, S-adenosyl-L-homocysteine (4), and the most potent inhibitor reported to date, SK&F 64139 (7), was used to identify the residues involved in inhibitor binding. Four of these residues, Val53, Lys57, Glu219 and Asp267, were replaced, in turn, with alanine. All variants had increased Km values for phenylethanolamine (10), but only D267A showed a noteworthy (20-fold) decrease in its kcat value. Both WT hPNMT and D267A had similar kcat values for a rigid analogue, anti-9-amino-6-(trifluoromethyl)benzonorbornene (12), suggesting that Asp267 plays an important role in positioning the substrate but does not participate directly in catalysis. The Ki values for the binding of inhibitors such as 7 to the E219A and D267A variants increased by 2-3 orders of magnitude. Further, the inhibitors were shown to bind up to 50-fold more tightly in the presence of S-adenosyl-L-methionine (3), suggesting that the binding of the latter brings about a conformational change in the enzyme.

2-Hydroxyphenethylamine↗
PubMed2005

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking.

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 A r.m.s. deviation of Calpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.

Algorithms↗
PubMed2005

The structure of human SULT1A1 crystallized with estradiol. An insight into active site plasticity and substrate inhibition with multi-ring substrates.

Human SULT1A1 belongs to the supergene family of sulfotransferases (SULTs) involved in the sulfonation of xeno- and endobiotics. The enzyme is also one of the SULTs responsible for metabolic activation of mutagenic and carcinogenic compounds and therefore is implicated in various cancer forms. Further, it is not well understood how substrate inhibition takes place with rigid fused multiring substrates such as 17beta-estradiol (E2) at high substrate concentrations when subcellular fractions or recombinant enzymes are used. To investigate how estradiol binds to SULT1A1, we co-crystallized SULT1A1 with sulfated estradiol and the cofactor product, PAP (3'-phosphoadenosine 5'-phosphate). The crystal structure of SULT1A1 that we present here has PAP and one molecule of E2 bound in a nonproductive mode in the active site. The structure reveals how the SULT1A1 binding site undergoes conformational changes to accept fused ring substrates such as steroids. In agreement with previous reports, the enzyme shows partial substrate inhibition at high concentrations of E2. A model to explain these kinetics is developed based on the formation of an enzyme x PAP x E2 dead-end complex during catalysis. This model provides a very good quantitative description of the rate versus the [E2] curve. This dead-end complex is proposed to be that described by the observed structure, where E2 is bound in a nonproductive mode.

Adenosine Diphosphate↗
PubMed2005

Post-crystallization treatments for improving diffraction quality of protein crystals.

X-ray crystallography is the most powerful method for determining the three-dimensional structure of biological macromolecules. One of the major obstacles in the process is the production of high-quality crystals for structure determination. All too often, crystals are produced that are of poor quality and are unsuitable for diffraction studies. This review provides a compilation of post-crystallization methods that can convert poorly diffracting crystals into data-quality crystals. Protocols for annealing, dehydration, soaking and cross-linking are outlined and examples of some spectacular changes in crystal quality are provided. The protocols are easily incorporated into the structure-determination pipeline and a practical guide is provided that shows how and when to use the different post-crystallization treatments for improving crystal quality.

Cross-Linking Reagents↗
PubMed2005

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active.

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.

Binding Sites↗
PubMed2005

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system.

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.

Animals↗
PubMed2005

Older schizophrenia patients have more disrupted sleep and circadian rhythms than age-matched comparison subjects.

Patient reports and laboratory studies suggest schizophrenia patients have disrupted sleep across age groups. Studies have not compared overall sleep/wake patterns or circadian (24-h) activity rhythms of older community dwelling schizophrenia patients to matched comparison subjects. This study examined whether older schizophrenia patients had more disrupted sleep/wake patterns and circadian activity rhythms than age- and gender-matched normal comparison subjects (NCS). Twenty-eight older schizophrenia patients and 28 age- and gender-matched NCS were studied with three days of continuous wrist actigraphy. Nighttime and daytime actigraphically estimated sleep and wake, circadian activity rhythms and light exposure patterns were compared with and without years of education as a covariate. Patients spent longer in bed, had more disrupted nighttime sleep, slept more during the day, and had less robust circadian rhythms of activity and light exposure compared to NCS. Differences persisted in education-adjusted analyses. Within patients, working was associated with improved sleep and circadian rhythms. Findings suggest the sleep and circadian rhythm disruption of older schizophrenia patients was more extensive than that of matched NCS suggesting patients' sleep disruption was above and beyond what is attributable to advanced age alone. A need exists to develop multicomponent interventions to address sleep difficulties specific to older schizophrenia patients.

Adult↗
PubMed2005

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relationship to cystatins and the tumor suppressor TIG1.

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Amino Acid Motifs↗
PubMed2005

Nighttime oxygen desaturation and symptoms of sleep-disordered breathing in long-stay nursing home residents.

BACKGROUND: Sleep-disordered breathing (SDB) is common in older adults and has been implicated as a cause of decreased quality of life and even death. Sparse data exist on SDB in the nursing home setting. The authors evaluated SDB (using attended nocturnal pulse oximetry) in nursing home residents with daytime sleepiness and nighttime sleep disturbance. METHODS: Pulse oximetry was used to estimate the prevalence of nighttime oxygen desaturation in 109 long-stay nursing home residents (mean [standard deviation] age = 86.2 [9.2] years; 74% women). Pulse oximetry findings were compared to a structured observational measurement of symptoms of SDB, the Observational Sleep Assessment Instrument. Seventy-one participants had concurrent wrist actigraphy to estimate total sleep time during oximetry recording. RESULTS: Using the oxygen desaturation index (ODI; average number of oxygen desaturations 4% or more below the baseline level per hour), the authors found that 40% of the residents had abnormal ODI (ODI more than 5, which is suggestive of SDB). Of all observational variables assessed, only loud breathing during sleep was significantly correlated with ODI (r =.284; p =.003). When ODI was adjusted for estimated total sleep time, higher adjusted ODI was associated with higher body mass index (kg/m(2)). CONCLUSIONS: Abnormal ODI is common in nursing home residents. Observed loud breathing at night and high body mass index may suggest that further assessment of SDB is indicated. Future research should determine the importance of SDB and abnormal nocturnal oxygen desaturation on functioning and quality of life in nursing home residents.

Aged↗
PubMed2005

Randomized, controlled trial of a nonpharmacological intervention to improve abnormal sleep/wake patterns in nursing home residents.

OBJECTIVES: Abnormal sleep/wake patterns are common in nursing home residents. Lifestyle and environmental factors likely contribute to these poor sleep patterns. The objective of this study was to test a multidimensional, nonpharmacological intervention to improve abnormal sleep/wake patterns in nursing home residents. DESIGN: Randomized, controlled trial. SETTING: Four nursing homes in the Los Angeles area. PARTICIPANTS: Residents were screened for excessive daytime sleeping (asleep > or = 15% of daytime observations) and nighttime sleep disruption (asleep < 80% of nighttime hours, according to wrist actigraphy). Four hundred ninety-two residents were screened; 339 had excessive daytime sleeping. Of these, 133 had nighttime sleep disruption and consented to participate; 120 completed baseline assessments, and 118 (77% female, mean age 86.9, 90% non-Hispanic white) were randomized to intervention versus usual care. INTERVENTION: Five consecutive days and nights of efforts to decrease daytime in-bed time, 30 minutes or more of daily sunlight exposure, increased physical activity, structured bedtime routine, and efforts to decrease nighttime noise and light. MEASUREMENTS: Seventy-two consecutive hours of wrist actigraphy (nighttime sleep) and structured behavioral observations (daytime sleep and participation in social and physical activities and social conversation) at baseline and repeated at follow-up while the intervention or usual care condition was in place. RESULTS: The only effect on nighttime sleep was a modest decrease in mean duration of nighttime awakenings in intervention participants (10.6 minutes at baseline, 9.8 minutes at follow-up) versus an increase in controls (9.8 minutes at baseline, 13.8 minutes at follow-up) (F=4.27, P=.04). There were no significant effects on percentage of nighttime sleep or number of nighttime awakenings. There was a significant decrease in daytime sleeping in intervention participants (32% of daytime observations asleep at baseline, 21% at follow-up), with no change in controls (32% at baseline, 30% at follow-up; F=20.68, P<.001). Intervention participants had increased participation in social (F=22.42, P<.001) and physical (F=12.65, P=.001) activities and social conversation (F=5.04, P=.03). CONCLUSION: A multidimensional, nonpharmacological intervention into lifestyle and environmental factors that likely contribute to abnormal sleep/wake patterns in nursing home residents resulted in decreased daytime sleeping and increased participation in social and physical activities and social conversation. Nonpharmacological interventions should be considered in the management of abnormal sleep/wake patterns in nursing home residents. The main effect may be a significant decrease in daytime sleeping, which may translate to an improvement in quality of life.

Aged↗
PubMed2005

Depression in older patients admitted for postacute nursing home rehabilitation.

OBJECTIVES: To describe the prevalence, recognition, and persistence of depression in older adults undergoing postacute rehabilitation in a nursing home (NH) setting and to explore the effect of depression on rehabilitation outcomes. DESIGN: Prospective cohort study. SETTING: One rehabilitative NH in the Los Angeles area. PARTICIPANTS: One hundred fifty-eight patients (aged >/=65) admitted for postacute rehabilitation over a 9-month recruitment period. MEASUREMENTS: Depression was assessed using the 15-item Geriatric Depression Scale (GDS-15) or the Cornell Scale for Depression (in participants with dementia). Medical records were reviewed for documentation of depression and antidepressant use before and during the rehabilitative NH stay. Rehabilitation process was assessed using total amount of successfully completed therapy (minutes). Rehabilitation outcome was assessed using the motor component of the Functional Independence Measure (mFIM). Measures were performed at admission and 2 months later. RESULTS: Of the 646 potentially eligible patients admitted during the study, 158 consented, and 151 were screened for depression. Forty-two (27.8%) had depressive symptoms (GDS=6 or Cornell=5). Of these, only 15 had a documented diagnosis of depression, and 12 were receiving antidepressants. Depression was associated with longer NH stay but not with discharge mFIM score. Two months later, depression persisted in 24 participants and was associated with worse mFIM (55.5+/-22.7 vs 67.0+/-23.7, depressed vs nondepressed; P=.03). CONCLUSION: Depression was common, underrecognized, and undertreated in these postacute rehabilitation patients. Depression generally persisted and was associated with worse functional status at 2-month follow-up.

Acute Disease↗
PubMed2004

Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide.

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria. DsbG, a member of this family, has disulfide bond isomerase and chaperone activity. Here, we present two crystal structures of DsbG at 1.7and 2.0-A resolution that are meant to represent the reduced and oxidized forms, respectively. The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form. This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm. We propose that a Thr residue that is conserved in the cis-Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process. Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding. Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces. This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins.

Amino Acid Sequence↗
PubMed2004

Countering cooperative effects in protease inhibitors using constrained beta-strand-mimicking templates in focused combinatorial libraries.

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

Combinatorial Chemistry Techniques↗
PubMed2004

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3.

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site.

Arylsulfotransferase↗
PubMed2004

Molecular recognition of sub-micromolar inhibitors by the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase.

The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.

Benzazepines↗
PubMed2004

The timing of activity rhythms in patients with dementia is related to survival.

BACKGROUND: Older adults with dementia often have disruptions in circadian rhythms, including disruptions of the rest-activity rhythm. These disruptions are a product of internal neuronal activity and external environmental influences, both of which are deficient in dementia. However, the consequences of disturbed rhythms are unknown. This study examined the relationship between rest-activity rhythms and death in patients with dementia. METHODS: The authors recruited 149 older adults with dementia (104 women; mean age, 84.1 years) from nursing homes. Activity was recorded with wrist actigraphs from each participant for 3 days. Survival was determined by examining public death records. Cox proportional hazards models were used to determine which aspects of rest-activity rhythms were related to survival. RESULTS: The timing of each participant's rest-activity rhythm compared with a sample of persons without dementia was related to survival, such that those who more closely resembled the persons without dementia lived longer. CONCLUSIONS: Although rest-activity rhythms as a whole were not related to survival, the timing of the rhythm was. Patients with dementia appear to develop an abnormal timing of their rhythms, which is predictive of shorter survival. It may be possible to intervene with these patients to correct the timing of their rhythms and possibly prolong their lives.

Aged↗
PubMed2004

Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid.

In high concentrations of fresh nonimmune human serum, Mycobacterium tuberculosis activates the alternative pathway of complement and binds C3 protein, resulting in enhanced phagocytosis by complement receptors on human alveolar macrophages. Yet in the lung, the alternative pathway of complement is relatively inactive compared to the classical pathway. To begin to determine whether C3 opsonophagocytosis of M. tuberculosis by alveolar macrophages can occur in the lung of the immunologically naive host, we characterized the binding of C3 to M. tuberculosis in different concentrations of fresh nonimmune human serum and concentrated human bronchoalveolar lavage fluid. Here we show that in human serum, C3 binding to M. tuberculosis is rapid, initiated by either the alternative pathway or the classical pathway, depending on the concentration of serum, and occurs by covalent linkages between the bacterial surface and the C3 cleavage products, C3b or C3bi. Human bronchoalveolar lavage fluid contains C3 protein and functional classical pathway activity that mediates the binding of C3 to the surface of M. tuberculosis. These studies provide evidence that when M. tuberculosis is first inhaled into the lungs of the human host, the bacterium is opsonized by C3 cleavage via classical pathway activation within the alveolus, providing a C3-dependent entry pathway into resident alveolar macrophages.

Bronchoalveolar Lavage Fluid↗