Neurogranin modulates the rate of association between calmodulin and target peptides.

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25-1.5 µM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses.

Disulfide bonds play a crucial role in folding and structural stabilization of monoclonal antibodies (mAbs). Disulfide bond reduction may happen during the mAb manufacturing process, resulting in low molecular weight species and possible failure to meet product specifications. Although many mitigation strategies have been developed to prevent disulfide reduction, to the best of our knowledge, reforming disulfide bonds from the reduced antibody in manufacturing has not previously been reported. Here, we explored a novel rescue strategy in the downstream process to repair the broken disulfide bonds via in-vitro redox reactions on Protein A resin. Redox conditions including redox pair (cysteine/cystine ratio), pH, temperature, and reaction time were examined to achieve high antibody purity and a high reaction rate. Under the optimal redox condition, >90% reduced antibody could be reoxidized to form an intact antibody on Protein A resin in an hour. In addition, this study showed high flexibility on the range of the intact mAb fraction in the initial reduced mAb sample (the lower limit of intact mAb faction could be 14% based on the data reported in this study). Furthermore, a kinetic model based on elementary oxidative reactions was constructed to help optimize the reoxidation conditions and to predict product purity. Together, the deep understanding of interchain disulfide bond reoxidation, combined with the predictive kinetic model, provided a good foundation to implement a rescue strategy to generate high-purity antibodies with substantial cost savings in manufacturing processes.

Conformational frustration in calmodulin-target recognition.

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations.

Protein recognition and selection through conformational and mutually induced fit.

Protein-protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, mechanistic insights were gained in defining the pathways leading to recognition and in how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.

A single-institution experience of hand surgery litigation in a major replantation center.

BACKGROUND: Bellevue Hospital Medical Center is a level 1 trauma center in New York and a major referral center for complex hand injuries and amputations. These injuries typically occur at the workplace and are thought to be highly litiginous in nature. This study was conducted to analyze the cases involving hand surgery litigation related to trauma over the last 8 years at this institution. METHODS: The authors performed a retrospective chart review of all claims filed against Bellevue Hospital Medical Center after treatment for a hand injury during 2001 to 2009. Twenty-three patients in total were identified and reviewed for age, mechanism/type of injury, complications, decision to replant, average time after injury to post claim, and whether settlement was obtained. RESULTS: One of 23 patients who filed suit against Bellevue Hospital Medical Center received a successful settlement involving an incident surrounding the loss of a nonreplantable part. Of 168 patients in whom 219 replantations/revascularizations were performed, five patients filed claims, all surrounding a failed attempt. In total, there were seven complications: five failed replants, one failed thenar flap, and one patient who needed a revision completion amputation. CONCLUSIONS: The majority of the patients who filed claims did so because of the decision not to replant. Only 2.98 percent (five of 168) of all attempted revascularization/replantation patients filed claims against the authors' institution; all claims were notably dropped. The legal system appears to support physicians and institutions that treat these complex injuries. Better patient understanding of the decision-making process and complications involving treatment of traumatic hand injuries may decrease the number of future lawsuits.

Correlation of levels and patterns of genomic instability with histological grading of DCIS.

BACKGROUND: Histological grading of ductal carcinoma-in-situ (DCIS) lesions separates DCIS into three subgroups (well-, moderately, or poorly differentiated). It is unclear, however, whether breast disease progresses along a histological continuum or whether each grade represents a separate disease. In this study, levels and patterns of allelic imbalance (AI) were examined in DCIS lesions to develop molecular models that can distinguish pathological classifications of DCIS. METHODS: Laser microdissected DNA samples were collected from DCIS lesions characterized by a single pathologist including well- (n = 18), moderately (n = 35), and poorly differentiated (n = 47) lesions. A panel of 52 microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer was used to assess patterns of AI. RESULTS: The overall frequency of AI increased significantly (P < .001) with increasing grade (well differentiated, 12%; moderately differentiated, 17%; poorly differentiated, 26%). Levels of AI were not significantly different between well- and moderately differentiated grades of disease but were significantly higher (P < .0001) in poorly differentiated compared with well- or moderately differentiated disease. No statistically significant differences in patterns of AI were detected between well- and moderately differentiated disease; however, AI occurred significantly more frequently (P < .05) in high-grade lesions at chromosomes 6q25-q27, 8q24, 9p21, 13q14, and 17p13.1, and significantly more frequently in low-grade lesions at chromosome 16q22.3-q24.3. CONCLUSIONS: The inability to discriminate DCIS at the genetic level suggests that grades 1 and 2 DCIS may represent a single, non-high-grade form of DCIS, whereas poorly differentiated DCIS seems to be a genetically more advanced disease that may represent a discrete disease entity, characterized by a unique spectrum of genetic alterations.

Timing of critical genetic changes in human breast disease.

BACKGROUND: Breast cancer development has been characterized as a nonobligatory sequence of histological changes from normal epithelium through invasive malignancy. Although genetic alterations are thought to accumulate stochastically during tumorigenesis, little is known about the timing of critical mutations. This study examined allelic imbalance (AI) in tissue samples representing a continuum of breast cancer development to examine the evolution of genomic instability. METHODS: Laser-microdissected DNA samples were collected from histologically normal breast specimens (n = 25), atypical ductal hyperplasia (ADH, n = 16), ductal carcinoma-in-situ (DCIS, n = 37), and stage I to III invasive carcinomas (n = 72). Fifty-two microsatellite markers representing 26 chromosomal regions commonly deleted in breast cancer were used to assess patterns of AI. RESULTS: AI frequencies were <5% in histologically normal and ADH specimens, 20% in DCIS lesions, and approximately 25% in invasive tumors. Mann-Whitney tests showed (1) that levels of AI in ADH samples did not differ significantly from those in histologically normal tissues and (2) that AI frequencies in DCIS lesions were not significantly different from those in invasive carcinomas. ADH and DCIS samples, however, differed significantly (P < .0001). CONCLUSIONS: DCIS lesions contain levels of genomic instability that are characteristic of advanced invasive tumors, and this suggests that the biology of a developing carcinoma may already be predetermined by the in situ stage. Observations that levels of AI in ADH lesions are similar to those in disease-free tissues provide a genomic rationale for why prevention strategies at the ADH level are successful and why cases with ADH involving surgical margins do not require further resection.