Biomedical subjects

Roman Sakowicz

Publications and source records attributed to Roman Sakowicz.

6 recordsLinked to original sources

PubMed2004

Mechanism of inhibition of human KSP by monastrol: insights from kinetic analysis and the effect of ionic strength on KSP inhibition.

Kinesin motor proteins utilize the energy from ATP hydrolysis to transport cellular cargo along microtubules. Kinesins that play essential roles in the mechanics of mitosis are attractive targets for novel antimitotic cancer therapies. Monastrol, a cell-permeable inhibitor that specifically inhibits the kinesin Eg5, the Xenopus laevis homologue of human KSP, can cause mitotic arrest and monopolar spindle formation. In this study, we show that the extent of monastrol inhibition of KSP microtubule-stimulated ATP hydrolysis is highly dependent upon ionic strength. Detailed kinetic analysis of KSP inhibition by monastrol in the presence and absence of microtubules suggests that monastrol binds to the KSP-ADP complex, forming a KSP-ADP-monastrol ternary complex, which cannot bind to microtubules productively and cannot undergo further ATP-driven conformational changes.

Adenosine Diphosphate↗

PubMed2004

Antitumor activity of a kinesin inhibitor.

Several members of the kinesin family of microtubule motor proteins play essential roles in mitotic spindle function and are potential targets for the discovery of novel antimitotic cancer therapies. KSP, also known as HsEg5, is a kinesin that plays an essential role in formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. We identified a potent inhibitor of KSP, CK0106023, which causes mitotic arrest and growth inhibition in several human tumor cell lines. Here we show that CK0106023 is an allosteric inhibitor of KSP motor domain ATPase with a Ki of 12 nM. Among five kinesins tested, CK0106023 was specific for KSP. In tumor-bearing mice, CK0106023 exhibited antitumor activity comparable to or exceeding that of paclitaxel and caused the formation of monopolar mitotic figures identical to those produced in cultured cells. KSP was most abundant in proliferating human tissues and was absent from cultured postmitotic neurons. These findings are the first to demonstrate the feasibility of targeting mitotic kinesins for the treatment of cancer.

Adenosine Triphosphatases↗

PubMed2004

Structure of a kinesin microtubule depolymerization machine.

With their ability to depolymerize microtubules (MTs), KinI kinesins are the rogue members of the kinesin family. Here we present the 1.6 A crystal structure of a KinI motor core from Plasmodium falciparum, which is sufficient for depolymerization in vitro. Unlike all published kinesin structures to date, nucleotide is not present, and there are noticeable differences in loop regions L6 and L10 (the plus-end tip), L2 and L8 and in switch II (L11 and helix4); otherwise, the pKinI structure is very similar to previous kinesin structures. KinI-conserved amino acids were mutated to alanine, and studied for their effects on depolymerization and ATP hydrolysis. Notably, mutation of three residues in L2 appears to primarily affect depolymerization, rather than general MT binding or ATP hydrolysis. The results of this study confirm the suspected importance of loop 2 for KinI function, and provide evidence that KinI is specialized to hydrolyze ATP after initiating depolymerization.

Animals↗

PubMed2003

Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice.

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice-stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.

Adenosine Triphosphatases↗

PubMed2002

A mechanism for microtubule depolymerization by KinI kinesins.

Whereas most kinesins motor along microtubules, KinI kinesins are microtubule depolymerizing machines. Surprisingly, we found that a KinI fragment consisting of only the motor core is capable of ATP-dependent depolymerization. The motor binds along microtubules in all nucleotide states, but in the presence of AMPPNP, microtubule depolymerization also occurs. Structural characterization of the products of AMPPNP-induced destabilization revealed a snapshot of the disassembly machine in action as it precisely deformed a tubulin dimer. While conventional kinesins use the energy of ATP binding to execute a "powerstroke," KinIs use it to bend the underlying protofilament. Thus, the relatively small class-specific differences within the KinI motor core modulate a fundamentally conserved mode of interaction with microtubules to produce a unique depolymerizing activity.

Adenosine Triphosphate↗

PubMed1999

Adociasulfates 1-6, Inhibitors of Kinesin Motor Proteins from the Sponge Haliclona (aka Adocia) sp.

Adociasulfates 1-6 (1-6) were isolated from an extract of the Palauan sponge Haliclona (aka Adocia) sp. that inhibited the transport of stabilized microtubules by the motor protein kinesin, which was immobilized on a microscope slide. The structures of adociasulfates 1-6, the relative stereochemistry of adociasulfates 1, 2, 5, and 6, and the relative stereochemistry of subunits of adociasulfates 3 and 4 were determined by interpretation of spectroscopic data. In a quantitative assay that measures ATP hydrolysis by kinesin, adociasulfates 2 and 6 were the most active.

Journal Article↗