Antibody association in solution: cluster distributions and mechanisms.

Understanding factors that affect the clustering and association of antibodies molecules in solution is critical to their development as therapeutics. For 19 different monoclonal antibody (mAb) solutions, we measured the viscosities, the second virial coefficients, the Kirkwood-Buff integrals, and the cluster distributions of the antibody molecules as functions of protein concentration. Solutions were modeled using the statistical-physics Wertheim liquid-solution theory, representing antibodies as Y-shaped molecular structures of seven beads each. We found that high-viscosity solutions result from more antibody molecules per cluster. Multi-body properties such as viscosity are well predicted experimentally by the 2-body Kirkwood-Buff quantity, G22, but not by the second virial coefficient, B22, and well-predicted theoretically from the Wertheim protein-protein sticking energy. Weakly interacting antibodies are rate-limited by nucleation; strongly interacting ones by propagation. This approach gives a way to relate micro to macro properties of solutions of associating proteins.

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlin-null skeletal muscle.

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J. Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft.

Repeated repeat problems: Combinatorial effect of C9orf72-derived dipeptide repeat proteins.

A microsatellite expansion mutation in C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). The expansion mutation leads to C9orf72 loss of function, RNA foci formation, and generation of five species of non-AUG RAN translated dipeptide repeat proteins (DPRs), such as poly(GA), poly(GP), poly(GR), poly(PA), and poly(PR). Although one cell can contain more than type of DPRs, information about interplay between different DPR species is limited. Here we show that the combined expression of distinct C9orf72-derived dipeptide repeat species produces cellular outcomes and structural differences that are unique compared to the expression of a single DPR species, suggesting the complex biological interactions that occur when multiple DPR variants are simultaneously expressed. Our data highlights the need for further analysis of how combined expression of different DPRs affects the disease state.

Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262.

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu2+, Mg2+, and Fe2+. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

Carbonyl-based blue autofluorescence of proteins and amino acids.

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF's sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA.

When Good Goes Awry: The Aggregation of Protein Therapeutics.

Protein therapeutics are playing an increasingly important role in treatment of a variety of human diseases. However, like the rest of proteins, they are susceptible to aggregation. Aggregation of proteinaceous pharmaceuticals can cause a loss of efficacy and, potentially, cytotoxicity and an immunogenic response. This review describes various ways protein therapeutics aggregate and a variety of approaches taken to prevent or minimize this process.

Effects of osmolytes on solvent features of water in aqueous solutions.

The solvatochromic solvent features of water (dipolarity/polarizability, π*, hydrogen bond donor acidity, α, and hydrogen bond acceptor basicity, ß) of water have been determined in aqueous solutions of erythritol, glucose, inositol, sarcosine, xylitol and urea with concentrations from 0 to ~3 M and higher. The concentration effects of the osmolytes on the solvent features of water were characterized and compared with those reported previously for sorbitol, sucrose, trimethylamine N-oxide (TMAO), and trehalose. The solvent features of water in solutions of all osmolytes except TMAO and sarcosine were established to be linearly interrelated. It is shown that the concentration effects of essentially all nonionic osmolytes depend on osmolytes' lipophilicity, molecular polarizability, and polar surface area. It is demonstrated that solubility of various compounds in aqueous solutions of glucose, sucrose, sorbitol, and urea of varied concentrations may be described in terms of solvent dipolarity/polarizability of water in these solutions. Surface tension of aqueous solutions of sucrose and sorbitol may also be described in the same terms. The relative permittivity of aqueous solutions of glucose and sucrose may be described in terms of the solvent hydrogen bond donor acidity of water. It is suggested that the effects of nonionic osmolytes on behavior of proteins and nucleic acids in aqueous media may be considered in terms of the altered solvent features of water instead of "nano-molecular crowding" effect.

Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.

Intrinsic disorder in proteins involved in amyotrophic lateral sclerosis.

Five structurally and functionally different proteins, an enzyme superoxide dismutase 1 (SOD1), a TAR-DNA binding protein-43 (TDP-43), an RNA-binding protein FUS, a cofilin-binding protein C9orf72, and polypeptides generated as a result of its intronic hexanucleotide expansions, and to lesser degree actin-binding profilin-1 (PFN1), are considered to be the major drivers of amyotrophic lateral sclerosis. One of the features common to these proteins is the presence of significant levels of intrinsic disorder. The goal of this study is to consider these neurodegeneration-related proteins from the intrinsic disorder perspective. To this end, we employed a broad set of computational tools for intrinsic disorder analysis and conducted intensive literature search to gain information on the structural peculiarities of SOD1, TDP-43, FUS, C9orf72, and PFN1 and their intrinsic disorder predispositions, and the roles of intrinsic disorder in their normal and pathological functions.

The unfolding pathways of the native and molten globule states of 5-aminolevulinate synthase.

In this communication, we report the equilibrium and kinetic properties of the unfolding pathways of the native (pH 7.5) and alkaline molten globule (pH 10.5) states of the pyridoxal 5'-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS). The stability of the molten globule state is adversely affected by thermal- and guanidine hydrochloride (GuHCl)-induced denaturation, and the equilibrium unfolding pathways, irrespective of pH, cannot be described with simple two-state models. Rapid kinetic measurements, in the presence of denaturing GuHCl concentrations, reveal that at pH 10.5, the rate of ALAS denaturation is 3 times faster than at pH 7.5. From pH jump experiments, comparable rates for the denaturation of the tertiary structure and PLP-microenvironment were discerned, indicating that the catalytic active site geometry strongly depends on the stable tertiary structural organization. Lastly, we demonstrate that partially folded ALAS tends to self-associate into higher oligomeric species at moderate GuHCl concentrations.

Structural differences between amyloid beta oligomers.

In Alzheimer's disease, soluble Aß oligomers are believed to play important roles in the disease pathogenesis, and their levels correlate with cognitive impairment. We have previously shown that Aß oligomers can be categorized into multiple structural classes based on their reactivity with conformation-dependent antibodies. In this study, we analyzed the structures of Aß40 oligomers belonging to two of these classes: fibrillar and prefibrillar oligomers. We found that fibrillar oligomers were similar in structure to fibrils but were less stable towards denaturation while prefibrillar oligomers were found to be partially disordered. These results are consistent with previously proposed structures for both oligomer classes while providing additional structural information.

Mechanistic study of the inhibitory activity of Geum urbanum extract against α-Synuclein fibrillation.

The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's disease and is hypothesized to be linked to disease development, although this is not yet conclusive. Lewy bodies and Lewy neurites primarily consist of fibrillated α-Synuclein; yet, there is no treatment available targeting stabilization of α-Synuclein in its native state. The aim of the present study was to investigate the inhibitory activity of an ethanolic extract of Geum urbanum against α-Synuclein fibrillation and examine the structural changes of α-Synuclein in the presence of the extract. The anti-fibrillation and anti-aggregation activities of the plant extract were monitored by thioflavin T fibrillation assays and size exclusion chromatography, while structural changes were followed by circular dichroism, Fourier transform infrared spectroscopy, intrinsic fluorescence, small angle X-ray scattering and electron microscopy. Since the extract is a complex mixture, structure-function relationships could not be determined. Under the experimental conditions investigated, Geum urbanum was found to inhibit α-Synuclein fibrillation in a concentration dependent way, and to partly disintegrate preformed α-Synuclein fibrils. Based on the structural changes of α-Synuclein in the presence of extract, we propose that Geum urbanum delays α-Synuclein fibrillation either by reducing the fibrillation ability of one or more of the aggregation prone intermediates or by directing α-Synuclein aggregation towards a non-fibrillar state. However, whether these alterations of the fibrillation pathway lead to less pathogenic species is yet to be determined.

α-Lactalbumin: Of Camels and Cows.

Since camel milk has been attributed with various medicinal properties not found in bovine milk, we are systematically examining the differences between different proteins in bovine and camel milk. The purpose of this study is to investigate the structural differences between the bovine and camel α- lactalbumins. α-Lactalbumin is a highly abundant protein present in the milk of all mammalian species. Here we found several structural differences between bovine and camel α-lactalbumins: camel protein is more stable towards thermal and pHmediated denaturation but less stable towards guanidine hydrochloride-mediated unfolding, aggregates faster and is predicted to be more disordered than bovine α- lactalbumin.

Disorder in Milk Proteins: α-Lactalbumin. Part C. Peculiarities of Metal Binding.

This is a concluding part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. In this paper, we describe the peculiarities of metal binding to a multifunctional milk protein, α-lactalbumin, which has two domains, a large α-helical domain and a small ß-sheet domain connected by a calcium binding loop. It is known that in addition to four disulfide bonds, the native fold of this protein is stabilized by binding of a calcium ion. In fact, although in various mammals, α-lactalbumins are rather poorly conserved possessing the overall sequence identity of ~16%, the positions of all eight cysteines and a calcium binding site (residues DKFLDDDITDDI in human protein) are strongly conserved. Curiously, this conserved calcium binding loop is located within a region with increased structural flexibility. Besides canonical calcium binding, α-lactalbumin is known to interact with other metals, such as zinc (for which it has a specific binding site), and, in its apo-form, it can bind other divalent and monovalent cations. The binding of Mg2+, Na+, and K+ to the Ca2+ site increases α-lactalbumin stability against action of heat and various denaturing agents, with the higher stabilization effects being imposed by the stronger bound metal ions.

Disorder in Milk Proteins: α -Lactalbumin. Part A. Structural Properties and Conformational Behavior.

This is a first part of the two-part article that continues a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We introduce here α-lactalbumin, a small (Mr 14 200), simple, acidic (pI 4-5), Ca(2+)-binding protein that might constitute up to 20% of total milk protein. Although function (it is one of the two components of lactose synthase that catalyzes the final step of the lactose biosynthesis in the lactating mammary gland), structure (protein has two domains, a large α -helical domain and a small ß -sheet domain connected by a calcium binding loop), and folding mechanisms (α-lactalbumin is well-known as a classic example of the molten globule state) of this model globular protein are relatively well understood, α-lactalbumin continues to surprise researchers and clearly continues to have high discovery potential. The goal of this review is to summarize some recent advances in the field of α-lactalbumin research and to analyze the peculiarities of the "intrinsic disorder code" of this protein.

Disorder in Milk Proteins: α-Lactalbumin. Part B. A Multifunctional Whey Protein Acting as an Oligomeric Molten Globular "Oil Container" in the Anti-Tumorigenic Drugs, Liprotides.

This is a second part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We continue to describe α-lactalbumin, a small globular Ca2+-binding protein, which besides being one of the two components of lactose synthase that catalyzes the final step of the lactose biosynthesis in the lactating mammary gland, possesses a multitude of other functions. In fact, recent studies indicated that some partially folded forms of this protein possess noticeable bactericidal activity and other forms might be related to induction of the apoptosis of tumor cells. In its anti-tumorigenic function, oligomeric α-lactalbumin serves as a founding member of a new family of anticancer drugs termed liprotides (for lipids and partially denatured proteins), where an oligomeric molten globular protein acts as an "oil container" or cargo for the delivery of oleic acid to the cell membranes.

Murine erythroid 5-aminolevulinate synthase: Truncation of a disordered N-terminal extension is not detrimental for catalysis.

5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme, catalyzes the initial step of heme biosynthesis in non-plant eukaryotes. The precursor form of the enzyme is translated in the cytosol, and upon mitochondrial import, the N-terminal targeting presequence is proteolytically cleaved to generate mature ALAS. In bone marrow-derived erythroid cells, a mitochondrial- and site-specific endoprotease of yet unknown primary structure, produces a protein shorter than mature erythroid ALAS (ALAS2) found in peripheral blood erythroid cells. This truncated ALAS2 lacks the presequence and the N-terminal sequence (corresponding to ~7 KDa molecular mass) present in ALAS2 from peripheral blood erythroid cells. How the truncation affects the structural topology and catalytic properties of ALAS2 is presently not known. To address this question, we created a recombinant, truncated, murine ALAS2 (ΔmALAS2) devoid of the cleavable N-terminal region and examined its catalytic and biophysical properties. The N-terminal truncation of mALAS2 did not significantly affect the organization of the secondary structure, but a subtle reduction in the rigidity of the tertiary structure was noted. Furthermore, thermal denaturation studies revealed a decrease of 4.3°C in the Tm value of ΔmALAS2, implicating lower thermal stability. While the kcat of ΔmALAS2 is slightly increased over that of the wild-type enzyme, the slowest step in the ΔmALAS2-catalyzed reaction remains dominated by ALA release. Importantly, intrinsic disorder algorithms imply that the N-terminal region of mALAS2 is highly disordered, and thus susceptible to proteolysis. We propose that the N-terminal truncation offers a cell-specific ALAS2 regulatory mechanism without hindering heme synthesis.

Mechanistic binding insights for 1-deoxy-D-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax.

We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Pseudocatalytic Antiaggregation Activity of Antibodies: Immunoglobulins can Influence α-Synuclein Aggregation at Substoichiometric Concentrations.

Protein aggregation is involved in a variety of diseases. Alteration of the aggregation pathway, either to produce less toxic structures or to increase aggregate clearance, is a promising therapeutic route. Both active and passive immunization has been used for this purpose. However, the mechanism of action of antibodies on protein aggregates is not completely clear especially given poor ability of antibodies to cross blood-brain barrier. Here, we have shown that antibodies can interfere with protein aggregation at substoichiometric concentrations (as low as 1:1000 antibody to protein ratio). This is an indication that antibodies interact with aggregation intermediates in chaperone-like manner altering the aggregation pathways at very low antibody levels. This observation supports earlier suggestions that antibodies can inhibit aggregation by interaction with low abundance aggregation intermediates.